Oxadiazoline ligands for modulating the expression of exogenous genes via an ecdysone receptor complex

ABSTRACT

The present invention relates to methods to use non-steroidal ligands in nuclear receptor-based inducible gene expression system to modulate exogenous gene expression in which an ecdysone receptor complex comprising: a DNA binding domain; a ligand binding domain; a transactivation domain; and a ligand is contacted with a DNA construct comprising: the exogenous gene and a response element; wherein the exogenous gene is under the control of the response element and binding of the DNA binding domain to the response element in the presence of the ligand results in activation or suppression of the gene.

This application claims priority to U.S. provisional application No.60/449,467 filed Feb. 21, 2003.

FIELD OF THE INVENTION

This invention relates to the field of biotechnology or geneticengineering. Specifically, this invention relates to the field of geneexpression. More specifically, this invention relates to non-steroidalligands for natural and mutated nuclear receptors and their use in anuclear receptor-based inducible gene expression system and methods ofmodulating the expression of a gene within a host cell using theseligands and inducible gene expression system.

BACKGROUND OF THE INVENTION

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. However, the citation ofany reference herein should not be construed as an admission that suchreference is available as “Prior Art” to the instant application.

In the field of genetic engineering, precise control of gene expressionis a valuable tool for studying, manipulating, and controllingdevelopment and other physiological processes. Gene expression is acomplex biological process involving a number of specificprotein-protein interactions. In order for gene expression to betriggered, such that it produces the RNA necessary as the first step inprotein synthesis, a transcriptional activator must be brought intoproximity of a promoter that controls gene transcription. Typically, thetranscriptional activator itself is associated with a protein that hasat least one DNA binding domain that binds to DNA binding sites presentin the promoter regions of genes. Thus, for gene expression to occur, aprotein comprising a DNA binding domain and a transactivation domainlocated at an appropriate distance from the DNA binding domain must bebrought into the correct position in the promoter region of the gene.

The traditional transgenic approach utilizes a cell-type specificpromoter to drive the expression of a designed transgene. A DNAconstruct containing the transgene is first incorporated into a hostgenome. When triggered by a transcriptional activator, expression of thetransgene occurs in a given cell type.

Another means to regulate expression of foreign genes in cells isthrough inducible promoters. Examples of the use of such induciblepromoters include the PR1-a promoter, prokaryotic repressor-operatorsystems, immunosuppressive-immunophilin systems, and higher eukaryotictranscription activation systems such as steroid hormone receptorsystems and are described below.

The PR1-a promoter from tobacco is induced during the systemic acquiredresistance response following pathogen attack. The use of PR1-a may belimited because it often responds to endogenous materials and externalfactors such as pathogens, UV-B radiation, and pollutants. Generegulation systems based on promoters induced by heat shock, interferonand heavy metals have been described (Wurn et al., 1986, Proc. Natl.Acad. Sci. USA 83:5414-5418; Arnheiter et al., 1990 Cell 62:51-61;Filmus et al., 1992 Nucleic Acids Research 20:27550-27560). However,these systems have limitations due to their effect on expression ofnon-target genes. These systems are also leaky.

Prokaryotic repressor-operator systems utilize bacterial repressorproteins and the unique operator DNA sequences to which they bind. Boththe tetracycline (“Tet”) and lactose (“Lac”) repressor-operator systemsfrom the bacterium Escherichia colt have been used in plants and animalsto control gene expression. In the Tet system, tetracycline binds to theTetR repressor protein, resulting in a conformational change thatreleases the repressor protein from the operator which as a resultallows transcription to occur. In the Lac system, a lac operon isactivated in response to the presence of lactose, or synthetic analogssuch as isopropyl-b-D-thiogalactoside. Unfortunately, the use of suchsystems is restricted by unstable chemistry of the ligands, i.e.tetracycline and lactose, their toxicity, their natural presence, or therelatively high levels required for induction or repression. For similarreasons, utility of such systems in animals is limited.

Immunosuppressive molecules such as FK506, rapamycin and cyclosporine Acan bind to immunophilins FKBP12, cyclophilin, etc. Using thisinformation, a general strategy has been devised to bring together anytwo proteins simply by placing FK506 on each of the two proteins or byplacing FK506 on one and cyclosporine A on another one. A synthetichomodimer of FK506 (FK1012) or a compound resulted from fusion ofFK506-cyclosporine (FKCsA) can then be used to induce dimerization ofthese molecules (Spencer et al., 1993, Science 262:1019-24; Belshaw etal., 1996 Proc Natl Acad Sci USA 93:4604-7). Gal4 DNA binding domainfused to FKBP12 and VP16 activator domain fused to cyclophilin, andFKCsA compound were used to show heterodimerization and activation of areporter gene under the control of a promoter containing Gal4 bindingsites. Unfortunately, this system includes immunosuppressants that canhave unwanted side effects and therefore, limits its use for variousmammalian gene switch applications.

Higher eukaryotic transcription activation systems such as steroidhormone receptor systems have also been employed. Steroid hormonereceptors are members of the nuclear receptor superfamily and are foundin vertebrate and invertebrate cells. Unfortunately, use of steroidalcompounds that activate the receptors for the regulation of geneexpression, particularly in plants and mammals, is limited due to theirinvolvement in many other natural biological pathways in such organisms.In order to overcome such difficulties, an alternative system has beendeveloped using insect ecdysone receptors (EcR).

Growth, molting, and development in insects are regulated by theecdysone steroid hormone (molting hormone) and the juvenile hormones(Dhadialla, et al., 1998. Annu. Rev. Entomol. 43: 545-569). Themolecular target for ecdysone in insects consists of at least ecdysonereceptor (EcR) and ultraspiracle protein (USP). EcR is a member of thenuclear steroid receptor super family that is characterized by signatureDNA and ligand binding domains, and an activation domain (Koelle et al.1991, Cell, 67:59-77). EcR receptors are responsive to a number ofsteroidal compounds such as ponasterone A and muristerone A. Recently,non-steroidal compounds with ecdysteroid agonist activity have beendescribed, including the commercially available insecticidestebufenozide and methoxyfenozide that are marketed world wide by Rohmand Haas Company (see International Patent Application No.PCT/EP96/00686 and U.S. Pat. No. 5,530,028). Both analogs haveexceptional safety profiles to other organisms.

The insect ecdysone receptor (EcR) heterodimerizes with Ultraspiracle(USP), the insect homologue of the mammalian RXR, and binds ecdysteroidsand ecdysone receptor response elements and activate transcription ofecdysone responsive genes. The EcR/USP/ligand complexes play importantroles during insect development and reproduction. The EcR is a member ofthe steroid hormone receptor superfamily and has five modular domains,A/B (transactivation), C (DNA binding, heterodimerization)), D (Hinge,heterodimerization), E (ligand binding, heterodimerization andtransactivation and F (transactivation) domains. Some of these domainssuch as A/B, C and E retain their function when they are fused to otherproteins.

Tightly regulated inducible gene expression systems or “gene switches”are useful for various applications such as gene therapy, large scaleproduction of proteins in cells, cell based high throughput screeningassays, functional genomics and regulation of traits in transgenicplants and animals.

The first version of EcR-based gene switch used Drosophila melanogasterEcR (DmEcR) and Mus musculus RXR (MmRXR) and showed that these receptorsin the presence of steroid, ponasterone A, transactivate reporter genesin mammalian cell lines and transgenic mice (Christopherson K. S., MarkM. R., Baja J. V., Godowski P. J. 1992, Proc. Natl. Acad. Sci. U.S.A.89: 6314-6318; No D., Yao T. P., Evans R. M., 1996, Proc. Natl. Acad.Sci. U.S.A. 93: 3346-3351). Later, Suhr et al. 1998, Proc. Natl. Acad.Sci. 95:7999-8004 showed that non-steroidal ecdysone agonist,tebufenozide, induced high level of transactivation of reporter genes inmammalian cells through Bombyx mori EcR (BmEcR) in the absence ofexogenous heterodimer partner.

International Patent Applications No. PCT/JS97/05330 (WO 97/38117) andPCT/US99/08381 (WO99/58155) disclose methods for modulating theexpression of an exogenous gene in which a DNA construct comprising theexogenous gene and an ecdysone response element is activated by a secondDNA construct comprising an ecdysone receptor that, in the presence of aligand therefor, and optionally in the presence of a receptor capable ofacting as a silent partner, binds to the ecdysone response element toinduce gene expression. The ecdysone receptor of choice was isolatedfrom Drosophila melanogaster. Typically, such systems require thepresence of the silent partner, preferably retinoid X receptor (RXR), inorder to provide optimum activation. In mammalian cells, insect ecdysonereceptor (FcR) heterodimerizes with retinoid X receptor (RXR) andregulates expression of target genes in a ligand dependent manner.International Patent Application No. PCT/US98/14215 (WO 99/02683)discloses that the ecdysone receptor isolated from the silk moth Bombyxmori is functional in mammalian systems without the need for anexogenous dimer partner.

U.S. Pat. No. 6,265,173 B1 discloses that various members of thesteroid/thyroid superfamily of receptors can combine with Drosophilamelanogaster ultraspiracle receptor (USP) or fragments thereofcomprising at least the dimerization domain of USP for use in a geneexpression system. U.S. Pat. No. 5,880,333 discloses a Drosophilamelanogaster FcR and ultraspiracle (USP) heterodimer system used inplants in which the transactivation domain and the DNA binding domainare positioned on two different hybrid proteins. Unfortunately, theseUSP-based systems are constitutive in animal cells and therefore, arenot effective for regulating reporter gene expression.

In each of these cases, the transactivation domain and the DNA bindingdomain (either as native EcR as in International Patent Application No.PCT/US98/14215 or as modified EcR as in International Patent ApplicationNo. PCT/US97/05330) were incorporated into a single molecule and theother heterodimeric partners, either USP or RXR, were used in theirnative state.

Drawbacks of the above described EcR-based gene regulation systemsinclude a considerable background activity in the absence of ligands andnon-applicability of these systems for use in both plants and animals(see U.S. Pat. No. 5,880,333). Therefore, a need exists in the art forimproved EcR-based systems to precisely modulate the expression ofexogenous genes in both plants and animals. Such improved systems wouldbe useful for applications such as gene therapy, large-scale productionof proteins and antibodies, cell-based high throughput screening assays,functional genomics and regulation of traits in transgenic animals. Forcertain applications such as gene therapy, it may be desirable to havean inducible gene expression system that responds well to syntheticnon-steroid ligands and at the same is insensitive to the naturalsteroids. Thus, improved systems that are simple, compact, and dependenton ligands that are relatively inexpensive, readily available, and oflow toxicity to the host would prove useful for regulating biologicalsystems.

Recently, it has been shown that an ecdysone receptor-based induciblegene expression system in which the transactivation and DNA bindingdomains are separated from each other by placing them on two differentproteins results in greatly reduced background activity in the absenceof a ligand and significantly increased activity over background in thepresence of a ligand (pending application PCT/US01/09050, incorporatedherein in its entirety by reference). This two-hybrid system is asignificantly improved inducible gene expression modulation systemcompared to the two systems disclosed in applications PCT/US97/05330 andPCT/US98/14215. The two-hybrid system exploits the ability of a pair ofinteracting proteins to bring the transcription activation domain into amore favorable position relative to the DNA binding domain such thatwhen the DNA binding domain binds to the DNA binding site on the gene,the transactivation domain more effectively activates the promoter (see,for example, U.S. Pat. No. 5,283,173). Briefly, the two-hybrid geneexpression system comprises two gene expression cassettes; the firstencoding a DNA binding domain fused to a nuclear receptor polypeptide,and the second encoding a transactivation domain fused to a differentnuclear receptor polypeptide. In the presence of ligand, the interactionof the first polypeptide with the second polypeptide effectively tethersthe DNA binding domain to the transactivation domain. Since the DNAbinding and transactivation domains reside on two different molecules,the background activity in the absence of ligand is greatly reduced.

A two-hybrid system also provides improved sensitivity to non-steroidalligands for example, diacylhydrazines, when compared to steroidalligands for example, ponasterone A (“PonA”) or muristerone A (“MurA”).That is, when compared to steroids, the non-steroidal ligands providehigher activity at a lower concentration. In addition, sincetransactivation based on EcR gene switches is often cell-line dependent,it is easier to tailor switching systems to obtain maximumtransactivation capability for each application. Furthermore, thetwo-hybrid system avoids some side effects due to overexpression of RXRthat often occur when unmodified RXR is used as a switching partner. Ina preferred two-hybrid system, native DNA binding and transactivationdomains of EcR or RXR are eliminated and as a result, these hybridmolecules have less chance of interacting with other steroid hormonereceptors present in the cell resulting in reduced side effects.

With the improvement in ecdysone receptor-based gene regulation systemsthere is an increase in their use in various applications resulting inincreased demand for ligands with higher activity than those currentlyexist. U.S. Pat. No. 6,258,603 B1 (and patents cited therein) discloseddibenzoythydrazine ligands, however, a need exists for additionalligands with different structures and physicochemical properties. Wehave discovered novel non-diacylhydrazine ligands which have notpreviously been described or shown to have the ability to modulate theexpression of transgenes.

SUMMARY OF THE INVENTION

The present invention relates to non-steroidal ligands for use innuclear receptor-based inducible gene expression system, and methods ofmodulating the expression of a gene within a host cell using theseligands with nuclear receptor-based inducible gene expression systems.

Applicants' invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation system witha ligand of the present invention. Specifically, Applicants' inventionprovides a method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; b) introducinginto the host cell a gene expression cassette comprising i) a responseelement comprising a domain to which the DNA binding domain from thefirst hybrid polypeptide of the gene expression modulation system binds;ii) a promoter that is activated by the transactivation domain of thesecond hybrid polypeptide of the gene expression modulation system; andiii) a gene whose expression is to be modulated; and c) introducing intothe host cell a ligand; whereby upon introduction of the ligand into thehost cell, expression of the gene is modulated. Applicants' inventionalso provides a method of modulating the expression of a gene in a hostcell comprising a gene expression cassette comprising a response elementcomprising a domain to which the DNA binding domain from the firsthybrid polypeptide of the gene expression modulation system binds; apromoter that is activated by the transactivation domain of the secondhybrid polypeptide of the gene expression modulation system; and a genewhose expression is to be modulated; wherein the method comprises thesteps of: a) introducing into the host cell a gene expression modulationsystem according to the invention; and b) introducing into the host cella ligand; whereby upon introduction of the ligand into the host,expression of the gene is modulated.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have discovered novel ligands for natural and mutated nuclearreceptors. Thus, Applicants' invention provides a ligand for use withecdysone receptor-based inducible gene expression system useful formodulating expression of a gene of interest in a host cell. In aparticularly desirable embodiment, Applicants' invention provides aninducible gene expression system that has a reduced level of backgroundgene expression and responds to submicromolar concentrations ofnon-steroidal ligand. Thus, Applicants' novel ligands and inducible geneexpression system and its use in methods of modulating gene expressionin a host cell overcome the limitations of currently available inducibleexpression systems and provide the skilled artisan with an effectivemeans to control gene expression.

The present invention is useful for applications such as gene therapy,large scale production of proteins and antibodies, cell-based highthroughput screening assays, functional genomics, proteomics,metabolomics, and regulation of traits in transgenic organisms, wherecontrol of gene expression levels is desirable. An advantage ofApplicants' invention is that it provides a means to regulate geneexpression and to tailor expression levels to suit the user'srequirements.

The present invention pertains to compounds of the general formula:

wherein X and X′ are independently O or S;

R¹ is

-   -   a) H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl,        (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, or benzyloxy;    -   b) unsubstituted or substituted phenyl wherein the substituents        are independently 1 to 5H; halo; nitro; cyano; hydroxy; amino        (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;        (C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;        (C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;        (C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;        (C₂-C₆)alkenyl optionally substituted with halo, cyano,        (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally        substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;        (C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;        (C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl;        (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido (—CONR^(a)R^(b));        amido (—NR^(a)COR^(b)); alkoxycarbonylamino (—NR^(a)CO₂R^(b));        alkylaminocarbonylamino (—NR^(a)CONR^(b)R^(c)); mercapto;        (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido        (—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted or        substituted phenyl wherein the substituents are independently 1        to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when        two adjacent positions on the phenyl ring are substituted with        alkoxy groups, these groups, together with the carbon atoms to        which they are attached, may be joined as a linkage (—OCH₂O—) or        (—OCH₂CH₂O—) to form a 5- or 6-membered dioxolano or dioxano        heterocyclic ring;    -   c) unsubstituted or substituted naphthyl wherein the        substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino;    -   d) unsubstituted or substituted benzothiophene-2-yl,        benzothiophene-3-yl, benzofuran-2-yl, or benzofuran-3-yl wherein        the substituents are independently 1 to 3 halo, nitro, hydroxy,        (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or (C₁-C₆)alkoxycarbonyl        (—CO₂R^(a));    -   e) unsubstituted or substituted 2, 3, or 4-pyridyl wherein the        substituents are independently 1 to 3 halo, cyano, nitro,        hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or (C₁-C₆)haloalkoxy;    -   f) unsubstituted or substituted 5-membered heterocycle selected        from furyl, thiophenyl, triazolyl, pyrrolyl, isopyrroyl,        pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl, oxazolyl, or        isooxazolyl wherein the substituents are independently 1 to 3        halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,        (C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or        substituted phenyl wherein the substituents are independently 1        to 3 halo, nitro, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy,        (C₁-C₆)haloalkoxy, carboxy, (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)),        or amino (—NR^(a)R^(b));    -   g) aromatic-substituted or unsubstituted phenyl(C₁-C₈)alkyl,        phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein        the aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or    -   h) aromatic-substituted or unsubstituted phenylamino,        phenyl(C₁-C₆)alkylamino, or phenylcarbonylamino wherein the        aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino;

wherein R^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl, orphenyl;

R² and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)_(x)—), analkyloxylalkyl linkage (—(CH₂)_(y)—O—(CH₂)_(z)—), an alkylaminoalkyllinkage (—(CH₂)_(y)NR^(a)(CH₂)_(z)—), or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon atom towhich they are attached,

wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a) is H, (C₁-C₆)alkyl, orphenyl; and

R⁴ is unsubstituted or substituted phenyl wherein the substituents areindependently 1 to 5H; halo; nitro; cyano; hydroxy; amino(—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl;(C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy; (C₁-C₆)haloalkoxy;(C₁-C₆)alkoxy(C₁-C₆)alkyl; (C₁-C₆)alkoxy(C₁-C₆)alkoxy;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy(—OCOR^(a)); carboxamido (—CONR^(a)R^(b)); amido (—NR^(a)COR^(b));alkoxycarbonylamino (—NR^(a)CO₂R^(b)); alkylaminocarbonylamino(—NR^(a)CONR^(b)R^(c)); mercapto; (C₁-C₆)alkylthio;(C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido (—S(O)R^(a)); sulfamido(—SO₂NR^(b)R^(a)); or unsubstituted or substituted phenyl wherein thesubstituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, or amino; or when two adjacent positions on the phenylring are substituted with alkoxy groups, these groups, together with thecarbon atoms to which they are attached, may be joined to form a 5- or6-membered dioxolano (—OCH₂O—) or dioxano (—OCH₂CH₂O—) heterocyclicring; wherein R^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl,or phenyl;

provided that R⁴ is not 3-nitrophenyl or 4-nitrophenyl, and

when R⁴ is phenyl, then R¹ is not phenyl,

when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, or

when R⁴ is 4-chlorophenyl, then R¹ is not methyl.

Compounds of the general formula are preferred when:

X and X′ are independently O or S;

R¹ is

-   -   a) H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl,        (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, or benzyloxy;    -   b) unsubstituted or substituted phenyl wherein the substituents        are independently 1 to 5H; halo; nitro; cyano; hydroxy;        (C₁-C₆)alkyl; (C₁₋₆)haloalkyl; (C₁-C₆)cyanoalkyl;        (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; (C₁-C₆)haloalkoxy;        (C₁-C₆)alkoxy(C₁-C₆)alkyl; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;        (C₂-C₆)alkenyl optionally substituted with halo, cyano,        (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally        substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;        (C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;        (C₁-C₆)alkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a));        carboxamido (—CONR^(a)R^(b)); amido (—NR^(a)COR^(b));        (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido (—S(O)R^(a));        sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted or substituted        phenyl wherein the substituents are independently 1 to 3 halo,        nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two        adjacent positions on the phenyl ring are substituted with        alkoxy groups, these groups, together with the carbon atoms to        which they are attached, may be joined as a linkage (—OCH₂O—) or        (—OCH₂CH₂O—) to form a 5- or 6-membered dioxolano or dioxano        heterocyclic ring;    -   c) unsubstituted or substituted benzothiophene-2-yl, or        benzofuran-2-yl wherein the substituents are independently 1 to        3 halo, nitro, hydroxy, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy;    -   d) unsubstituted or substituted 2, 3, or 4-pyridyl wherein the        substituents are independently 1 to 3 halo, cyano, nitro,        hydroxy, (C₁-C₆)alkyl, (C₁-C₅)alkoxy, or (C₁-C₆)haloalkoxy;    -   e) unsubstituted or substituted 5-membered heterocycle selected        from furyl, thiophenyl, triazolyl, pyrazolyl, thiazolyl,        isothiazolyl, oxazolyl, or isooxazolyl wherein the substituents        are independently 1 to 3 halo, nitro, hydroxy, (C₁-C₆)alkyl,        (C₁-C₆)alkoxy, carboxy, (C₁-C₆)alkoxycarbonyl (—CO₂R), or        unsubstituted or substituted phenyl wherein the substituents are        independently 1 to 3 halo, nitro, (C₁-C₆)alkyl,        (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy, carboxy, or        (C₁-C₄)alkoxycarbonyl (—CO₂R^(a));    -   f) aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,        phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein        the aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, or (C₁-C₆)alkyl; or    -   g) aromatic-substituted or unsubstituted phenylamino,        phenyl(C₁-C₆)alkylamino, or phenylcarbonylamino wherein the        aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, or (C₁-C₆)alkyl;

wherein R^(a) and R^(b) are independently H, (C₁-C₆)alkyl, or phenyl;

R² and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)_(x)—), analkyloxylalkyl linkage (—(CH₂)_(y)O(CH₂)_(z)—), an alkylaminoalkyllinkage (—(CH₂)_(y)NR^(a)(CH₂)_(z)—), or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon atom towhich they are attached,

wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a) is H, (C₁-C₆)alkyl, orphenyl; and

R⁴ is unsubstituted or substituted phenyl wherein the substituents areindependently 1 to 5H; halo; nitro; cyano; hydroxy; (C₁-C₆)alkyl;(C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); (C₁-C₆)alkylsulfonyl;(C₁-C₆)alkylsulfoxido (—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); orunsubstituted or substituted phenyl wherein the substituents areindependently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino;or when two adjacent positions on the phenyl ring are substituted withalkoxy groups, these groups, together with the carbon atoms to whichthey are attached, may be joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—)to form a 5- or 6-membered dioxolano or dioxano heterocyclic ring;wherein R^(a) and R^(b) are independently H, (C₁-C₆)alkyl, or phenyl;

provided that R⁴ is not 3-nitrophenyl or 4-nitrophenyl, and

when R⁴ is phenyl, then R¹ is not phenyl,

when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, or

when R⁴ is 4-chlorophenyl, then R¹ is not methyl.

Compounds of the general formula are more preferred when:

X is O;

X′ is O or S;

R¹ is

-   -   a) H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, or        (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl;    -   b) unsubstituted or substituted phenyl wherein the substituents        are independently 1 to 5H; halo; nitro; cyano; (C₁-C₆)alkyl;        (C₁-C₆)haloalkyl; (C₁-C₆)alkoxy; (C₁-C₆)haloalkoxy;        (C₁-C₆)alkylcarbonyl; (C₁-C₆)alkoxycarbonyl;        carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); or phenyl;        or when two adjacent positions on the phenyl ring are        substituted with alkoxy groups, these groups, together with the        carbon atoms to which they are attached, may be joined as a        linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a 5- or 6-membered        dioxolano or dioxano heterocyclic ring;    -   c) unsubstituted or substituted benzothiophene-2-yl, or        benzofuran-2-yl wherein the substituents are independently 1 to        3 halo, nitro, hydroxy, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy;    -   d) unsubstituted or substituted furyl or thiophenyl wherein the        substituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkyl,        (C₁-C₆)alkoxy, carboxy, (C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or        phenyl;    -   e) aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,        phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein        the aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, or (C₁-C₆)alkyl; or    -   f) aromatic-substituted or unsubstituted phenylamino,        phenyl(C₁-C₆)alkylamino, or phenylcarbonylamino wherein the        aromatic substituents are independently 1 to 3 halo, nitro,        (C₁-C₆)alkoxy, or (C₁-C₆)alkyl;

wherein R^(a) and R^(b) are independently H, (C₁-C₆)alkyl, or phenyl;

R² and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)alkoxy(C₁-C₆)alkyl, phenyl, or together as an alkane linkage(—(CH₂)_(x)—), an alkyloxylalkyl linkage (—(CH₂)_(y)—O—(CH₂)_(z)—), analkylaminoalkyl linkage (—(CH₂)_(y)NR^(a)(CH₂)_(z)—), or analkylbenzoalkyl linkage (—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ringwith the carbon atom to which they are attached,

wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a) is H, (C₁-C₆)alkyl, orphenyl; and

R⁴ is unsubstituted or substituted phenyl wherein the substituents areindependently 1 to 5H; halo; nitro; cyano; (C₁-C₆)alkyl;(C₁-C₆)haloalkyl; (C₁-C₆)alkoxy; (C₁-C₆)haloalkoxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)alkoxycarbonyl; carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); or phenyl; or when twoadjacent positions on the phenyl ring are substituted with alkoxygroups, these groups, together with the carbon atoms to which they areattached, may be joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a5- or 6-membered dioxolano or dioxano heterocyclic ring; wherein R^(a)and R^(b) are independently H, (C₁-C₆)alkyl, or phenyl;

provided that R⁴ is not 3-nitrophenyl or 4-nitrophenyl, and

when R⁴ is phenyl, then R¹ is not phenyl,

when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, or

when R⁴ is 4-chlorophenyl, then R¹ is not methyl.

Compounds of the general formula are even more preferred when:

X and X′ are O;

R¹ is phenyl, 4-chlorophenyl-, 4-ethylphenyl-,2-ethyl-3,4-ethylenedioxyphenyl, 3-fluorophenyl-,2-fluoro-4-ethylphenyl-, 2-methyl-3-methoxyphenyl-,2-ethyl-3-methoxyphenyl, 3-methylphenyl-, 2-methoxyphenyl-,2-nitrophenyl-, 3-nitrophenyl-, 2-furanyl-, benzyl-,benzothiophene-2-yl-, phenylamino-, benzyloxymethyl, phenoxymethyl-,3-toluoylamino-, benzylamino-, benzoylamino-, ethoxycarbonylethyl-, or3-chloro-2,2,3,3-tetrafluoroethyl;

R² and R³ are independently methyl, ethyl, or together as atetramethylene (—(CH2)₄—), 4-pyrano (—CH₂CH₂OCH₂CH₂—), ormethylenebenzoethylene (—CH₂-1-benzo-2-CH₂CH₂—) linkage form a ring withthe carbon atom to which they are attached; and

R⁴ is phenyl, 4-biphenyl, 4-chlorophenyl, 2,4-dimethoxyphenyl,3,5-dimethylphenyl, 2-methoxyphenyl, 3,4-methylenedioxyphenyl,3-trifluoromethylphenyl, or 4-trifluoromethoxyphenyl;

provided that when R⁴ is phenyl, then R¹ is not phenyl.

The compounds of the present invention most preferred are the following:

Compound R1 R2 R3 R4 RG-120001 PhNHC(O)— CH₃— CH₃— 3-CF₃-Ph- RG-1200023-F-benzoyl CH₃— CH₃— 3,5-di-CH₃-Ph- RG-120003 2-furanoyl- —(CH₂)₄—3,5-di-CH₃-Ph- RG-120004 CClF₂CF₂C(O)— CH₃CH₂— CH₃— 3,5-di-CH₃-Ph-RG-120005 4-CH₃CH₂-benzoyl- —CH₂CH₂OCH₂CH₂— 3,5-di-CH₃-Ph- RG-120006PhCH₂OCH₂C(O)— CH₃— CH₃— 3-CF₃-Ph- RG-120008 2-CH₃CH₂-3-CH₃O-benzoyl-—CH₂CH₂OCH₂CH₂— 3,5-di-CH₃-Ph- RG-120009 PhCH₂OCH₂C(O)— —CH₂CH₂OCH₂CH₂—3,5-di-CH₃-Ph- RG-120011 Benzoyl- —(CH₂)₄— 3,5-di-CH₃-Ph- RG-1200122-furanoyl- CH₃— CH₃— 2-CH₃O-Ph- RG-120013 PhOCH₂C(O)— —CH₂CH₂OCH₂CH₂—Ph- RG-120014 CH₃CH₂OC(O)CH₂CH₂C(O)— —(CH₂)₄— Ph- RG-120015 Benzoyl-CH₃— CH₃— 3-CF₃-Ph- RG-120016 2-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃—2-CH₃O-Ph- RG-120017 PhNHC(O)— CH₃— CH₃— 3,4-OCH₂O-Ph- RG-120018PhCH₂OCH₂C(O)— CH₃— CH₃— 2-CH₃O-Ph- RG-120019 Benzoyl- —CH₂CH₂OCH₂CH₂—3,5-di-CH₃-Ph- RG-120020 2-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃— 4-Ph-Ph-RG-120021 PhCH₂C(O)— CH₃— CH₃— 3-CF₃-Ph- RG-1200222-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃— 4-CF₃O-Ph- RG-1200232-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃— 3,4-OCH₂O-Ph- RG-120024 4-Cl-benzoyl-CH₃— CH₃— 3,5-di-CH₃-Ph- RG-120025 PhNHC(O)— CH₃— CH₃— 2-CH₃O-Ph-RG-120026 4-CH₃CH₂-benzoyl- CH₃— CH₃— 2-CH₃O-Ph- RG-120027 PhNHC(O)——CH2CH2OCH2CH2— Ph- RG-120029 PhOCH₂C(O)— CH₃— CH₃— 4-CF₃O-Ph- RG-120030PhCH₂C(O)— —(CH₂)₄— Ph- RG-120031 CH3CH2OC(O)CH2CH2C(O)— —(CH₂)₄—3,5-di-CH₃-Ph- RG-120033 Benzoyl- CH₃— CH₃— 4-CF₃O-Ph- RG-120034PhCH₂OCH₂C(O)— —CH₂CH₂OCH₂CH₂— Ph- RG-120035 4-CH₃CH₂-benzoyl- CH₃— CH₃—4-Cl-Ph- RG-120037 2-CH₃-3-CH₃O-benzoyl- —CH₂-1-benzo-2-CH₂CH₂—3,5-di-CH₃-Ph- RG-120038 CH₃CH₂OC(O)CH₂CH₂C(O)— CH₃— CH₃—2,4-di-CH₃O-Ph- RG-120039 Benzoyl- CH₃CH₂— CH₃— 3,5-di-CH₃-Ph- RG-1200404-CH₃CH₂-benzoyl- —(CH₂)₄— 3,5-di-CH₃-Ph- RG-120041 PhOCH₂C(O)— —(CH₂)₄—3,5-di-CH₃-Ph- RG-120042 2-CH₃-3-CH₃O-benzoyl- CH₃— CH₃— Ph- RG-120044Benzoyl- —CH₂CH₂OCH₂CH₂— Ph- RG-120045 2-CH₃-3-CH₃O-benzoyl- CH₃— CH₃—3,5-di-CH₃-Ph- RG-120046 PhCH₂C(O)— —(CH₂)₄— 3,5-di-CH₃-Ph- RG-120047benzothiophene-2-C(O)— CH₃— CH₃— 2-CH₃O-Ph- RG-120048 PhOCH₂C(O)——CH₂CH₂OCH₂CH₂— 3,5-di-CH₃-Ph- RG-120049 2-CH₃CH₂-3-CH₃O-benzoyl-—(CH₂)₄— 3,5-di-CH₃-Ph- RG-120050 PhCH₂OCH₂C(O)— CH₃CH₂— CH₃—3,5-di-CH₃-Ph- RG-120051 PhNHC(O)— CH₃— CH₃CH₂— 3,5-di-CH₃-Ph- RG-120052PhCH₂OCH₂C(O)— —(CH₂)₄— 3,5-di-CH₃-Ph- RG-120054 PhNHC(O)——CH₂CH₂OCH₂CH₂— 3,5-di-CH₃-Ph- RG-120055 4-CH₃CH₂-benzoyl- CH₃—4-CF₃O-Ph- RG-120056 3-CH₃PhNHC(O)— CH₃— 3,5-di-CH₃-Ph- RG-120057PhOCH₂C(O)— CH₃— 2-CH₃O-Ph- RG-120058 2-CH₃CH₂-3-CH₃O-benzoyl- CH₃—2,4-di-CH₃O-Ph- RG-120059 CClF₂CF₂C(O)— CH₃— 3-CF₃-Ph- RG-1200604-CH₃CH₂-benzoyl- CH₃— 3,5-di-CH₃-Ph- RG-120061 4-CH₃CH₂-benzoyl- CH₃—3,4-OCH₂O-Ph- RG-120062 CClF₂CF₂C(O)— CH₃— 2-CH₃O-Ph- RG-120063CClF₂CF₂C(O)— —(CH₂)₄— Ph- RG-120066 PhCH₂OCH₂C(O)— CH₃— CH₃— 4-CF₃O-Ph-RG-120067 PhNHC(O)— CH₃— CH₃— 4-Cl-Ph- RG-1200692-CH₃CH₂-3-CH₃O-benzoyl- CH₃CH₂— CH₃— 3,5-di-CH₃-Ph- RG-1200702-furanoyl- CH₃— CH₃— 3-CF₃-Ph- RG-120071 2-furanoyl- —(CH₂)₄— Ph-RG-120072 PhNHC(O)— CH₃— CH₃— 3,5-di-CH₃-Ph- RG-120073 CClF₂CF₂C(O)—CH₃— CH₃— 4-Cl-Ph- RG-120075 2-CH₃O-benzoyl- CH₃— CH₃— 3,5-di-CH₃-Ph-RG-120076 2-CH₃CH₂-3-CH₃O-benzoyl- CH₃CH₂— CH₃— Ph- RG-1200772-CH₃-benzoyl- CH₃— CH₃— 3,5-di-CH₃-Ph- RG-120078 PhCH₂C(O)— CH₃— CH₃—2,4-di-CH₃O-Ph- RG-120079 PhOCH₂C(O)— CH₃— CH₃— 2,4-di-CH₃O-Ph-RG-120080 2-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃— 3,5-di-CH₃-Ph- RG-120081PhCH₂C(O)— CH₃— CH₃— 3,4-OCH₂O-Ph- RG-120082 2-furanoyl- CH₃— CH₃—4-Cl-Ph- RG-120083 CH₃CH₂OC(O)CH₂CH₂C(O)— CH₃— CH₃— 3,4-OCH₂O-Ph-RG-120084 PhCH₂C(O)— —CH₂CH₂OCH₂CH₂— 3,5-di-CH₃-Ph- RG-1200862-CH₃-3-CH₃O-benzoyl- —CH₂CH₂OCH₂CH₂— 3,5-di-CH₃-Ph- RG-120087benzothiophene-2-C(O)— CH₃— CH₃— 4-Cl-Ph- RG-120088 PhCH₂NHC(O)— CH₃—CH₃— 3,5-di-CH₃-Ph- RG-120089 Benzoyl- —(CH₂)₄— Ph- RG-120090CClF₂CF₂C(O)— —(CH₂)₄— 3,5-di-CH₃-Ph- RG-120091 3-NO₂-benzoyl- CH₃— CH₃—3,5-di-CH₃-Ph- RG-120092 2-CH₃CH₂-3-CH₃O-benzoyl- —(CH₂)₄— Ph- RG-1200932-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃— 3-CF₃-Ph- RG-120094 2-furanoyl- CH₃—CH₃— 4-CF₃O-Ph- RG-120095 PhNHC(O)— CH₃CH₂— CH₃— Ph- RG-120096 Benzoyl-CH₃— CH₃— 2,4-di-CH₃O-Ph- RG-120098 2-NO₂-benzoyl- CH₃— CH₃—3,5-di-CH₃-Ph- RG-120099 2-CH₃CH₂-3-CH₃O-benzoyl- CH₃— CH₃— 4-Cl-Ph-RG-120100 2-furanoyl- CH₃CH₂— CH₃— Ph- RG-120102 2-furanoyl- CH₃— CH₃—2,4-di-CH₃O-Ph- RG-120103 PhOCH₂C(O)— CH₃CH₂— CH₃— Ph- RG-1201062-furanoyl- CH₃CH₂— CH₃— 3,5-di-CH₃-Ph- RG-120108 benzothiophene-2-C(O)—CH₃— CH₃— 3-CF₃-Ph- RG-120109 benzothiophene-2-C(O)— CH₃— CH₃—4-CF₃O-Ph- RG-120110 PhCH₂OCH₂C(O)— CH₃— CH₃— 2,4-di-CH₃O-Ph- RG-120111PhC(O)NHC(O)— CH₃— CH₃— 3,5-di-CH₃-Ph- RG-120112 PhNHC(O)— —(CH₂)₄—3,5-di-CH₃-Ph- RG-120114 PhNHC(O)— CH₃— CH₃— 2,4-di-CH₃O-Ph- RG-1201154-CH₃CH₂-benzoyl- CH₃— CH₃— Ph- RG-120117 PhCH₂OCH₂C(O)— CH₃— CH₃—4-Cl-Ph- RG-120118 Benzoyl- CH₃CH₂— CH₃— Ph- RG-120120 PhCH₂C(O)—CH₃CH₂— CH₃— 3,5-di-CH₃-Ph- RG-120121 PhCH₂C(O)— CH₃— CH₃— 4-Cl-Ph-RG-120122 PhNHC(O)— CH₃— CH₃— 4-CF₃O-Ph- RG-120124 4-CH₃CH₂-benzoyl-—CH₂CH₂OCH₂CH₂— Ph- RG-120125 4-CH₃CH₂-benzoyl- —(CH₂)₄— Ph- RG-120126CH₃CH₂OC(O)CH₂CH₂C(O)— CH3CH₂— CH₃— 3,5-di-CH₃-Ph- RG-120127 PhOCH₂C(O)—CH₃— CH₃— 3,4-OCH₂O-Ph- RG-120128 4-CH₃CH₂-benzoyl- CH₃CH₂— CH₃—3,5-di-CH₃-Ph- RG-120129 benzothiophene-2-C(O)— CH₃— CH₃—2,4-di-CH₃O-Ph- RG-120130 PhCH₂C(O)— —CH₂CH₂OCH₂CH₂— Ph- RG-120132PhNHC(O)— —(CH₂)₄— Ph- RG-120133 benzothiophene-2-C(O)— CH₃CH₂— CH₃— Ph-RG-120135 4-CH₃CH₂-benzoyl- CH₃— CH₃— 2,4-di-CH₃O-Ph- RG-1201374-CH₃CH₂-benzoyl- CH₃CH₂— CH₃— Ph- RG-120138 2-furanoyl- —CH₂CH₂OCH₂CH₂—3,5-di-CH₃-Ph- RG-120140 benzothiophene-2-C(O)— CH₃— CH₃— 3,4-OCH₂O-Ph-RG-120141 PhOCH₂C(O)— CH₃CH₂— CH₃— 3,5-di-CH₃-Ph- RG-1201424-CH₃CH₂-benzoyl- CH₃— CH₃— 4-Ph-Ph- RG-120144 CH₃CH₂OC(O)CH₂CH₂C(O)—CH₃— CH₃— 2-CH₃O-Ph- RG-120145 PhCH₂OCH₂C(O)— CH₃— CH₃— 3,4-OCH₂O-Ph-RG-120146 PhCH₂C(O)— CH₃CH₂— CH₃— Ph- RG-120147 Benzoyl- CH₃— CH₃—2-CH₃O-Ph- RG-120148 4-CH₃CH₂-benzoyl- CH₃— CH₃— 3-CF₃-Ph- RG-1201492-furanoyl- CH₃— CH₃— 3,4-OCH₂O-Ph- RG-120150 benzothiophene-2-C(O)——(CH₂)₄— Ph- RG-120151 Benzoyl- CH₃— CH₃— 4-Cl-Ph- RG-120152benzothiophene-2-C(O)— —(CH₂)₄— 3,5-di-CH₃-Ph- RG-120153CH₃CH₂OC(O)CH₂CH₂C(O)— CH₃— CH₃— 3-CF₃-Ph- RG-120154 PhCH₂OCH₂C(O)—CH₃CH₂— CH₃— Ph- RG-120155 PhCH₂OCH₂C(O)— —(CH₂)₄— Ph- RG-120156Benzoyl- CH₃— CH₃— 3,4-OCH₂O-Ph- RG-120157 PhCH₂C(O)— CH₃— CH₃—2-CH₃O-Ph- RG-120158 PhOCH₂C(O)— —(CH₂)₄— Ph- RG-1201592-CH₃CH₂-3-CH₃O-benzoyl- —CH₂CH₂OCH₂CH₂— Ph- RG-120160 PhCH₂C(O)— CH₃—CH₃— 4-CF₃O-Ph- RG-120161 benzothiophene-2-C(O)— CH₃CH₂— CH₃—3,5-di-CH₃-Ph- RG-120162 PhOCH₂C(O)— CH₃— CH₃— 4-Cl-Ph- RG-120163PhOCH₂C(O)— CH₃— CH₃— 3-CF₃-Ph- RG-120164 CH₃CH₂OC(O)CH₂CH₂C(O)— CH₃—CH₃— 4-Cl-Ph- RG-121513 2-F-4-CH₃CH₂-benzoyl- CH₃— CH₃CH₂—3,5-di-CH₃-Ph- RG-121514 2-F-4-CH₃CH₂-benzoyl- —(CH₂)₄— Ph- RG-1215152-F-4-CH₃CH₂-benzoyl- —(CH₂)₄— 3,5-di-CH₃-Ph- RG-1215162-F-4-CH₃CH₂-benzoyl- CH₃— CH₃— Ph- RG-1215172-CH₃CH₂-3,4-OCH₂CH₂O-benzoyl- CH₃— CH₃— Ph- RG-1215182-CH₃CH2-3,4-OCH₂CH₂O-benzoyl- CH₃— CH₃CH₂— 3,5-di-CH₃-Ph-

Because the compounds of the general formula of the present inventionmay contain a number of stereogenic carbon atoms, the compounds mayexist as enantiomers, diastereomers, stereoisomers, or their mixtures,even if a stereogenic center is explicitly specified.

DEFINITIONS

The term “alkyl” includes both branched and straight chain alkyl groups.Typical alkyl groups include, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,isopentyl, n-hexyl, n-heptyl, isooctyl, nonyl, and decyl.

The term “halo” refers to fluoro, chloro, bromo or iodo.

The term “haloalkyl” refers to an alkyl group substituted with one ormore halo groups such as, for example, chloromethyl, 2-bromoethyl,3-iodopropyl, trifluoromethyl, and perfluoropropyl.

The term “cycloalkyl” refers to a cyclic aliphatic ring structure,optionally substituted with alkyl, hydroxy, or halo, such ascyclopropyl, methylcyclopropyl, cyclobutyl, 2-hydroxycyclopentyl,cyclohexyl, and 4-chlorocyclohexyl.

The term “hydroxyalkyl” refers to an alkyl group substituted with one ormore hydroxy groups such as, for example, hydroxymethyl and2,3-dihydroxybutyl.

The term “alkylsulfonyl” refers to a sulfonyl moiety substituted with analkyl group such as, for example, mesyl, and n-propylsulfonyl.

The term “alkenyl” refers to an ethylenically unsaturated hydrocarbongroup, straight or branched chain, having 1 or 2 ethylenic bonds suchas, for example, vinyl, allyl, 1-butenyl, 2-butenyl, isopropenyl, and2-pentenyl

The term “haloalkenyl” refers to an alkenyl group substituted with oneor more halo groups. The term “alkynyl” refers to an unsaturatedhydrocarbon group, straight or branched, having 1 or 2 acetylenic bondssuch as, for example, ethynyl and propargyl.

The term “alkylcarbonyl” refers to an alkylketo functionality, forexample acetyl, n-butyryl and the like.

The term “heterocyclyl” or “heterocycle” refers to an unsubstituted orsubstituted; saturated, partially unsaturated, or unsaturated 5 or6-membered ring containing one, two or three heteroatoms, preferably oneor two heteroatoms independently selected from the group consisting ofoxygen, nitrogen and sulfur. Examples of heterocyclyls include, forexample, pyridyl, thienyl, furyl, pyrimidinyl, pyrazinyl, quinolinyl,isoquinolinyl, pyrrolyl, indolyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, tetrahydropyranyl, morpholinyl, piperazinyl, dioxolanyl,and dioxanyl.

The term “alkoxy” includes both branched and straight chain alkyl groupsattached to a terminal oxygen atom. Typical alkoxy groups include, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, and tert-butoxy.

The term “haloalkoxy” refers to an alkoxy group substituted with one ormore halo groups such as, for example chloromethoxy, trifluoromethoxy,difluoromethoxy, and perfluoroisobutoxy.

The term “alkylthio” includes both branched and straight chain alkylgroups attached to a terminal sulfur atom such as, for examplemethylthio.

The term “haloalkylthio” refers to an alkylthio group substituted withone or more halo groups such as, for example trifluoromethylthio.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group such as, for example, isopropoxymethyl.

“Silica gel chromatography” refers to a purification method wherein achemical substance of interest is applied as a concentrated sample tothe top of a vertical column of silica gel or chemically-modified silicagel contained in a glass, plastic, or metal cylinder, and elution fromsuch column with a solvent or mixture of solvents.

“Flash chromatography” refers to silica gel chromatography performedunder air, argon, or nitrogen pressure typically in the range of 10 to50 psi.

“Gradient chromatography” refers to silica gel chromatography in whichthe chemical substance is eluted from a column with a progressivelychanging composition of a solvent mixture.

“Rf” is a thin layer chromatography term which refers to the fractionaldistance of movement of a chemical substance of interest on a thin layerchromatography plate, relative to the distance of movement of theeluting solvent system.

The term “isolated” for the purposes of the present invention designatesa biological material (nucleic acid or protein) that has been removedfrom its original environment (the environment in which it is naturallypresent). For example, a polynucleotide present in the natural state ina plant or an animal is not isolated, however the same polynucleotideseparated from the adjacent nucleic acids in which it is naturallypresent, is considered “isolated”. The term “purified” does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

A polynucleotide is in the “purified” state after purification of thestarting material or of the natural material by at least one order ofmagnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

A “nucleic acid” is a polymeric compound comprised of covalently linkedsubunits called nucleotides. Nucleic acid includes polyribonucleic acid(RNA) and polydeoxyribonucleic acid (DNA), both of which may besingle-stranded or double-stranded. DNA includes but is not limited tocDNA, genomic DNA, plasmids DNA, synthetic DNA, and semi-synthetic DNA.DNA may be linear, circular, or supercoiled.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6, 8, 9,10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51,54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleicacid according to the invention.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to an assembly of nucleotides that encode a polypeptide,and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to anucleic acid fragment that expresses a specific protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism, A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., 1989 infra). Hybridization andwashing conditions are well known and exemplified in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher T_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the instant invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In apreferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 63° C.; in an even more preferred embodiment,the T_(m) is 65-C.

Post-hybridization washes also determine stringency conditions. One setof preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 minutes (min), then repeated with2×SSC, 0.5% SDS at 45° C. for 30 minutes, and then repeated twice with0.2×SSC, 0.5% SDS at 50° C. for 30 minutes. A more preferred set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Anotherpreferred set of highly stringent conditions uses two final washes in0.1×SSC, 0.1% SDS at 65° C. Hybridization requires that the two nucleicacids comprise complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA, For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In a specific embodiment of the invention, polynucleotides are detectedby employing hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37 degrees Celsius, and a washingstep in 2×SSPE at least 63 degrees Celsius. In a preferred embodiment,the hybridization conditions comprise less than 200 mM salt and at least37 degrees Celsius for the hybridization step. In a more preferredembodiment, the hybridization conditions comprise 2×SSPE and 63 degreesCelsius for both the hybridization and washing steps.

In one embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferable a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 18 nucleotides, that is hybridizable to a genomicDNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin, has been covalentlyconjugated. A labeled oligonucleotide can be used as a probe to detectthe presence of a nucleic acid. Oligonucleotides (one or both of whichmay be labeled) can be used as PCR primers, either for cloning fulllength or a fragment of a nucleic acid, or to detect the presence of anucleic acid. An oligonucleotide can also be used to form a triple helixwith a DNA molecule. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A “primer” is an oligonucleotide that hybridizes to a target nucleicacid sequence to create a double stranded nucleic acid region that canserve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction.

“Polymerase chain reaction” is abbreviated PCR and means an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand means an in vitro method for enzymatically producing a target cDNAmolecule or molecules from an RNA molecule or molecules, followed byenzymatic amplification of a specific nucleic acid sequence or sequenceswithin the target cDNA molecule or molecules as described above. RT-PCRalso provides a means to detect the presence of the target molecule and,under quantitative or semi-quantitative conditions, to determine therelative amount of that target molecule within the starting pool ofnucleic acids.

A DNA “coding sequence” is a double-stranded DNA sequence that istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.“Suitable regulatory sequences” refer to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′(carboxyl)terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, andeven synthetic DNA sequences. If the coding sequence is intended forexpression in a eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and means a length of nucleicacid sequence, either DNA, cDNA or RNA, that comprises a translationstart signal or initiation codon, such as an ATG or AUG, and atermination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (← →) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→ ←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→ →)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5)to reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” refer toan enzyme that binds and cuts within a specific nucleotide sequencewithin double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” is any means for the cloning of and/or transfer of a nucleicacid into a host cell. A vector may be a replicon to which another DNAsegment may be attached so as to bring about the replication of theattached segment. A “replicon” is any genetic element (e.g., plasmid,phage, cosmid, chromosome, virus) that functions as an autonomous unitof DNA replication in vivo, i.e., capable of replication under its owncontrol. The term “vector” includes both viral and nonviral means forintroducing the nucleic acid into a cell in vitro, ex vivo or in vivo. Alarge number of vectors known in the art may be used to manipulatenucleic acids, incorporate response elements and promoters into genes,etc. Possible vectors include, for example, plasmids or modified virusesincluding, for example bacteriophages such as lambda derivatives, orplasmids such as pBR322 or pUC plasmid derivatives, or the Bluescriptvector. For example, the insertion of the DNA fragments corresponding toresponse elements and promoters into a suitable vector can beaccomplished by ligating the appropriate DNA fragments into a chosenvector that has complementary cohesive termini. Alternatively, the endsof the DNA molecules may be enzymatically modified or any site may beproduced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include but are notlimited to retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” is a “replicon”, which is a unit length of a nucleicacid, preferably DNA, that replicates sequentially and which comprisesan origin of replication, such as a plasmid, phage or cosmid, to whichanother nucleic acid segment may be attached so as to bring about thereplication of the attached segment. Cloning vectors may be capable ofreplication in one cell type and expression in another (“shuttlevector”).

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; and Hartmutet al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., 1987, PNAS 84:7413; Mackey, et al., 1988. Proc. Natl.Acad. Sci. U.S.A. 85:8027-8031; and Ulmer et al., 1993, Science259:1745-1748). The use of cationic lipids may promote encapsulation ofnegatively charged nucleic acids, and also promote fusion withnegatively charged cell membranes (Felgner and Ringold, 1989, Science337: 387-388). Particularly useful lipid compounds and compositions fortransfer of nucleic acids are described in International PatentPublications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127.The use of lipofection to introduce exogenous genes into the specificorgans in vivo has certain practical advantages. Molecular targeting ofliposomes to specific cells represents one area of benefit. It is clearthat directing transfection to particular cell types would beparticularly preferred in a tissue with cellular heterogeneity, such aspancreas, liver, kidney, and the brain. Lipids may be chemically coupledto other molecules for the purpose of targeting (Mackey, et al., 1988,supra). Targeted peptides, e.g., hormones or neurotransmitters, andproteins such as antibodies, or non-peptide molecules could be coupledto liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther. 3: 147-154; and Wu and Wu, 1987, J. Biol.Chem. 262: 4429-4432).

The term “transfection” means the uptake of exogenous or heterologousRNA or DNA by a cell. A cell has been “transfected” by exogenous orheterologous RNA or DNA when such RNA or DNA has been introduced insidethe cell. A cell has been “transformed” by exogenous or heterologous RNAor DNA when the transfected RNA or DNA effects a phenotypic change. Thetransforming RNA or DNA can be integrated (covalently linked) intochromosomal DNA making up the genome of the cell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “genetic region” will refer to a region of a nucleic acidmolecule or a nucleotide sequence that comprises a gene encoding apolypeptide.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” means an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, calorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” means a nucleic acid encoding an identifyingfactor that is able to be identified based upon the reporter gene'seffect, wherein the effect is used to track the inheritance of a nucleicacid of interest, to identify a cell or organism that has inherited thenucleic acid of interest, and/or to measure gene expression induction ortranscription. Examples of reporter genes known and used in the artinclude: luciferase (Luc), green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. Promotersthat cause a gene to be expressed in a specific cell type are commonlyreferred to as “cell-specific promoters” or “tissue-specific promoters”.Promoters that cause a gene to be expressed at a specific stage ofdevelopment or cell differentiation are commonly referred to as“developmentally-specific promoters” or “cell differentiation-specificpromoters”. Promoters that are induced and cause a gene to be expressedfollowing exposure or treatment of the cell with an agent, biologicalmolecule, chemical, ligand, light, or the like that induces the promoterare commonly referred to as “inducible promoters” or “regulatablepromoters”. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined, DNAfragments of different lengths may have identical promoter activity.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

The term “response element” means one or more cis-acting DNA elementswhich confer responsiveness on a promoter mediated through interactionwith the DNA-binding domains of the first chimeric gene. This DNAelement may be either palindromic (perfect or imperfect) in its sequenceor composed of sequence motifs or half sites separated by a variablenumber of nucleotides. The half sites can be similar or identical andarranged as either direct or inverted repeats or as a single half siteor multimers of adjacent half sites in tandem. The response element maycomprise a minimal promoter isolated from different organisms dependingupon the nature of the cell or organism into which the response elementwill be incorporated. The DNA binding domain of the first hybrid proteinbinds, in the presence or absence of a ligand, to the DNA sequence of aresponse element to initiate or suppress transcription of downstreamgene(s) under the regulation of this response element. Examples of DNAsequences for response elements of the natural ecdysone receptor includeRRGG/TTCANTGAC/ACYY (see Cherbas L., et. al., (1991), Genes Dev. 5,120-131); AGGTCAN_((n))AGGTCA, where N_((n)) can be one or more spacernucleotides (see D'Avino P P., et. al., (1995), Mol. Cell. Endocrinol,113, 1-9); and GGGTTGAATGAATTT (see Antoniewski C., et. al., (1994).Mol. Cell. Biol. 14, 4465-4474).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette”, “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and an EcRbased system which in the presence of one or more ligands, modulates theexpression of a gene into which the response element and promoter areincorporated.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The plasmids or vectors according to the invention may further compriseat least one promoter suitable for driving expression of a gene in ahost cell. The term “expression vector” means a vector, plasmid orvehicle designed to enable the expression of an inserted nucleic acidsequence following transformation into the host. The cloned gene, i.e.,the inserted nucleic acid sequence, is usually placed under the controlof control elements such as a promoter, a minimal promoter, an enhancer,or the like. Initiation control regions or promoters, which are usefulto drive expression of a nucleic acid in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to: viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoter, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, IP_(L), IP^(R), V7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-, cauliflower mosaic virus 35S, CMV 35S minimal, cassayavein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase,stress inducible, rice tungro bacilliform virus, plant super-promoter,potato leucine aminopeptidase, nitrate reductase, mannopine synthase,nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters(useful for expression in plant cells); animal and mammalian promotersknown in the art include, but are not limited to, the SV40 early (SV40e)promoter region, the promoter contained in the 3′ long terminal repeat(LTR) of Rous sarcoma virus (RSV), the promoters of the EIA or majorlate promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus(CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase(TK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha(EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of themouse metallothionein-L promoter and transcriptional control regions,the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and thelike), the promoters of the intermediate filaments (desmin,neurofilaments, keratin, GFAP, and the like), the promoters oftherapeutic genes (of the MDR, CFTR or factor VIII type, and the like),pathogenesis or disease related-promoters, and promoters that exhibittissue specificity and have been utilized in transgenic animals, such asthe elastase I gene control region which is active in pancreatic acinarcells; insulin gene control region active in pancreatic beta cells,immunoglobulin gene control region active in lymphoid cells, mousemammary tumor virus control region active in testicular, breast,lymphoid and mast cells; albumin gene, Apo AI and Apo AII controlregions active in liver, alpha-fetoprotein gene control region active inliver, alpha 1-antitrypsin gene control region active in the liver,beta-globin gene control region active in myeloid cells, myelin basicprotein gene control region active in oligodendrocyte cells in thebrain, myosin light chain-2 gene control region active in skeletalmuscle, and gonadotropic releasing hormone gene control region active inthe hypothalamus, pyruvate kinase promoter, villin promoter, promoter ofthe fatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like,

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a preferred embodiment ofthe invention, the termination control region may be comprise or bederived from a synthetic sequence, synthetic polyadenylation signal, anSV40 late polyadenylation signal, an SV40 polyadenylation signal, abovine growth hormone (BGH) polyadenylation signal, viral terminatorsequences, or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” means a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” is a regulatory regionthat is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

A “polypeptide” is a polymeric compound comprised of covalently linkedamino acid residues. Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup. A polypeptide of the invention preferably comprises at leastabout 14 amino acids.

A “protein” is a polypeptide that performs a structural or functionalrole in a living cell.

An “isolated polypeptide” or “isolated protein” is a polypeptide orprotein that is substantially free of those compounds that are normallyassociated therewith in its natural state (e.g., other proteins orpolypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is notmeant to exclude artificial or synthetic mixtures with other compounds,or the presence of impurities which do not interfere with biologicalactivity, and which may be present, for example, due to incompletepurification, addition of stabilizers, or compounding into apharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one (1) wild-type or naturally occurring amino acid with adifferent amino acid relative to the wild-type or naturally occurringpolypeptide. A substitution mutant polypeptide may comprise only one (1)wild-type or naturally occurring amino acid substitution and may bereferred to as a “point mutant” or a “single point mutant” polypeptide.Alternatively, a substitution mutant polypeptide may comprise asubstitution of two (2) or more wild-type or naturally occurring aminoacids with 2 or more amino acids relative to the wild-type or naturallyoccurring polypeptide. According to the invention, a Group H nuclearreceptor ligand binding domain polypeptide comprising a substitutionmutation comprises a substitution of at least one (1) wild-type ornaturally occurring amino acid with a different amino acid relative tothe wild-type or naturally occurring Group H nuclear receptor ligandbinding domain polypeptide.

Wherein the substitution mutant polypeptide comprises a substitution oftwo (2) or more wild-type or naturally occurring amino acids, thissubstitution may comprise either an equivalent number of wild-type ornaturally occurring amino acids deleted for the substitution, i.e., 2wild-type or naturally occurring amino acids replaced with 2non-wild-type or non-naturally occurring amino acids, or anon-equivalent number of wild-type amino acids deleted for thesubstitution, i.e., 2 wild-type amino acids replaced with 1non-wild-type amino acid (a substitution+deletion mutation), or 2wild-type amino acids replaced with 3 non-wild-type amino acids (asubstitution+insertion mutation).

Substitution mutants may be described using an abbreviated nomenclaturesystem to indicate the amino acid residue and number replaced within thereference polypeptide sequence and the new substituted amino acidresidue. For example, a substitution mutant in which the twentieth(20^(th)) amino acid residue of a polypeptide is substituted may beabbreviated as “x20z”, wherein “x” is the amino acid to be replaced,“20” is the amino acid residue position or number within thepolypeptide, and “z” is the new substituted amino acid. Therefore, asubstitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala”indicates that the mutant comprises an alanine residue (commonlyabbreviated in the art as “A” or “Ala”) in place of the glutamic acid(commonly abbreviated in the art as “E” or “Glu”) at position 20 of thepolypeptide.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253: 6551;Zoller and Smith, 1984, DNA 3: 479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83: 710),use of TAB® linkers (Pharmacia), restriction endonucleasedigestion/fragment deletion and substitution,PCR-mediated/oligonucleotide-directed mutagenesis, and the like.PCR-based techniques are preferred for site-directed mutagenesis (seeHiguchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology:Principles and Applications for DNA Amplification, H. Erlich, ed.,Stockton Press, Chapter 6, pp. 61-70).

“Fragment” of a polypeptide according to the invention will beunderstood to mean a polypeptide whose amino acid sequence is shorterthan that of the reference polypeptide and which comprises, over theentire portion with these reference polypeptides, an identical aminoacid sequence. Such fragments may, where appropriate, be included in alarger polypeptide of which they are a part. Such fragments of apolypeptide according to the invention may have a length of at least 2,3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30,35, 40, 45, 50, 100, 200, 240, or 300 amino acids.

A “variant” of a polypeptide or protein is any analogue, fragment,derivative, or mutant which is derived from a polypeptide or protein andwhich retains at least one biological property of the polypeptide orprotein. Different variants of the polypeptide or protein may exist innature. These variants may be allelic variations characterized bydifferences in the nucleotide sequences of the structural gene codingfor the protein, or may involve differential splicing orpost-translational modification. The skilled artisan can producevariants having single or multiple amino acid substitutions, deletions,additions, or replacements. These variants may include, inter alia; (a)variants in which one or more amino acid residues are substituted withconservative or non-conservative amino acids, (b) variants in which oneor more amino acids are added to the polypeptide or protein, (c)variants in which one or more of the amino acids includes a substituentgroup, and (d) variants in which the polypeptide or protein is fusedwith another polypeptide such as serum albumin. The techniques forobtaining these variants, including genetic (suppressions, deletions,mutations, etc.), chemical, and enzymatic techniques, are known topersons having ordinary skill in the art. A variant polypeptidepreferably comprises at least about 14 amino acids.

A “heterologous protein” refers to a protein not naturally produced inthe cell.

A “mature protein” refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or propeptides present in theprimary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-and propeptides still present, Pre- and propeptides may be but are notlimited to intracellular localization signals.

The term “signal peptide” refers to an amino terminal polypeptidepreceding the secreted mature protein. The signal peptide is cleavedfrom and is therefore not present in the mature protein. Signal peptideshave the function of directing and translocating secreted proteinsacross cell membranes. Signal peptide is also referred to as signalprotein.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., 1987, Cell 50:667.). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the instant application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., 1987, Cell 50: 667).

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., 1989, supra.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotide bases that donot substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary sequences. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the instant invention are those nucleic acid fragmentswhose DNA sequences are at least 70% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Preferred substantiallynucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are at least 80% identical to the DNAsequence of the nucleic acid fragments reported herein. More preferrednucleic acid fragments are at least 90% identical to the DNA sequence ofthe nucleic acid fragments reported herein. Even more preferred arenucleic acid fragments that are at least 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identity and/or isolate a nucleic acid fragment comprisingthe sequence,

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WNDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentially of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism. More preferably, thechange is at least 5-fold greater. Even more preferably, the change isat least 10-fold greater. Still more preferably, the change is at least100 fold greater. Even still more preferably, the change is at least500-fold greater. Ideally, modulation of each of the given systems byits respective ligand at a chosen concentration results in a measurablechange in the magnitude of expression of the gene of that system and nomeasurable change in expression of all other systems operable in thecell, tissue, or organism. In such cases the multiple inducible generegulation system is said to be “fully orthogonal”. The presentinvention is useful to search for orthogonal ligands and orthogonalreceptor-based gene expression systems such as those described inco-pending U.S. application Ser. No. 09/965,697, which is incorporatedherein by reference in its entirety.

The term “modulate” means the ability of a given ligand/receptor complexto induce or suppress the transactivation of an exogenous gene.

The term “exogenous gene” means a gene foreign to the subject, that is,a gene which is introduced into the subject through a transformationprocess, an unmutated version of an endogenous mutated gene or a mutatedversion of an endogenous unmutated gene. The method of transformation isnot critical to this invention and may be any method suitable for thesubject known to those in the art. For example, transgenic plants areobtained by regeneration from the transformed cells. Numeroustransformation procedures are known from the literature such asagroinfection using Agrobacterium tumefaciens or its T₁ plasmid,electroporation, microinjection of plant cells and protoplasts, andmicroprojectile transformation. Complementary techniques are known fortransformation of animal cells and regeneration of such transformedcells in transgenic animals. Exogenous genes can be either natural orsynthetic genes and therapeutic genes which are introduced into thesubject in the form of DNA or RNA which may function through a DNAintermediate such as by reverse transcriptase. Such genes can beintroduced into target cells, directly introduced into the subject, orindirectly introduced by the transfer of transformed cells into thesubject. The term “therapeutic gene” means a gene which imparts abeneficial function to the host cell in which such gene is expressed.Therapeutic genes are not naturally found in host cells.

The term “ecdysone receptor complex” generally refers to a heterodimericprotein complex consisting of two members of the steroid receptorfamily, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins(see Yao, T. P., et. al. (1993) Nature 366, 476-479; Yao, T.-P., et.al., (1992) Cell 71, 63-72). The functional ecdysteroid receptor complexmay also include additional protein(s) such as immunophilins. Additionalmembers of the steroid receptor family of proteins, known astranscriptional factors (such as DHR38, betaFTZ-1 or other insecthomologs), may also be ligand dependent or independent partners for EcRand/or USP. The ecdysone receptor complex can also be a heterodimer ofecdysone receptor protein and the vertebrate homolog of ultraspiracleprotein, retinoic acid-X-receptor (“RXR”) protein. Homodimer complexesof the ecdysone receptor protein or USP may also be functional undersome circumstances.

An ecdysteroid receptor complex can be activated by an activeecdysteroid or non-steroidal ligand bound to one of the proteins of thecomplex, inclusive of EcR, but not excluding other proteins of thecomplex.

The ecdysone receptor complex includes proteins which are members of thesteroid receptor superfamily wherein all members are characterized bythe presence of an amino-terminal transactivation domain, a DNA bindingdomain (“DBD”), and a ligand binding domain (“LBD”) separated by a hingeregion. Some members of the family may also have another transactivationdomain on the carboxy-terminal side of the LBD. The DBD is characterizedby the presence of two cysteine zinc fingers between which are two aminoacid motifs, the P-box and the D-box, which confer specificity forecdysone response elements. These domains may be either native,modified, or chimeras of different domains of heterologous receptorproteins.

The DNA sequences making up the exogenous gene, the response element,and the ecdysone receptor complex may be incorporated intoarchaebacteria, procaryotic cells such as Escherichia coli, Bacillussubtilis, or other enterobacteria, or eucaryptic cells such as plant oranimal cells. However, because many of the proteins expressed by thegene are processed incorrectly in bacteria, eucaryotic cells arepreferred. The cells may be in the form of single cells or multicellularorganisms. The nucleotide sequences for the exogenous gene, the responseelement, and the receptor complex can also be incorporated as RNAmolecules, preferably in the form of functional viral RNAs such astobacco mosaic virus. Of the eucaryotic cells, vertebrate cells arepreferred because they naturally lack the molecules which conferresponses to the ligands of this invention for the ecdysone receptor. Asa result, they are insensitive to the ligands of this invention. Thus,the ligands of this invention will have negligible physiological orother effects on transformed cells, or the whole organism. Therefore,cells can grow and express the desired product, substantially unaffectedby the presence of the ligand itself.

The term “subject” means an intact plant or animal or a cell from aplant or animal. It is also anticipated that the ligands will workequally well when the subject is a fungus or yeast. When the subject isan intact animal, preferably the animal is a vertebrate, most preferablya mammal.

The ligands of the present invention, when used with the ecdysonereceptor complex which in turn is bound to the response element linkedto an exogenous gene, provide the means for external temporal regulationof expression of the exogenous gene. The order in which the variouscomponents bind to each other, that is, ligand to receptor complex andreceptor complex to response element, is not critical. Typically,modulation of expression of the exogenous gene is in response to thebinding of the ecdysone receptor complex to a specific control, orregulatory, DNA element. The ecdysone receptor protein, like othermembers of the steroid receptor family, possesses at least threedomains, a transactivation domain, a DNA binding domain, and a ligandbinding domain. This receptor, like a subset of the steroid receptorfamily, also possesses less well-defined regions responsible forheterodimerization properties. Binding of the ligand to the ligandbinding domain of ecdysone receptor protein, after heterodimerizationwith USP or RXR protein, enables the DNA binding domains of theheterodimeric proteins to bind to the response element in an activatedform, thus resulting in expression or suppression of the exogenous gene.This mechanism does not exclude the potential for ligand binding toeither EcR or USP, and the resulting formation of active homodimercomplexes (e.g. EcR+EcR or USP+USP). Preferably, one or more of thereceptor domains can be varied producing a chimeric gene switch.Typically, one or more of the three domains may be chosen from a sourcedifferent than the source of the other domains so that the chimericreceptor is optimized in the chosen host cell or organism fortransactivating activity, complementary binding of the ligand, andrecognition of a specific response element. In addition, the responseelement itself can be modified or substituted with response elements forother DNA binding protein domains such as the GAL-4 protein from yeast(see Sadowski, et. al. (1988) Nature, 335, 563-564) or LexA protein fromE. coli (see Brent and Ptashne (1985), Cell, 43, 729-736) to accommodatechimeric ecdysone receptor complexes. Another advantage of chimericsystems is that they allow choice of a promoter used to drive theexogenous gene according to a desired end result. Such double controlcan be particularly important in areas of gene therapy, especially whencytotoxic proteins are produced, because both the timing of expressionas well as the cells wherein expression occurs can be controlled. Theterm “promoter” means a specific nucleotide sequence recognized by RNApolymerase. The sequence is the site at which transcription can bespecifically initiated under proper conditions. When exogenous genes,operatively linked to a suitable promoter, are introduced into the cellsof the subject, expression of the exogenous genes is controlled by thepresence of the ligand of this invention. Promoters may beconstitutively or inducibly regulated or may be tissue-specific (thatis, expressed only in a particular type of cell) or specific to certaindevelopmental stages of the organism.

Another aspect of this invention is a method to modulate the expressionof one or more exogenous genes in a subject, comprising administering tothe subject an effective amount, that is, the amount required to elicitthe desired gene expression or suppression, of a ligand comprising acompound of the present invention and wherein the cells of the subjectcontain:

a) an ecdysone receptor complex comprising:

-   -   1) a DNA binding domain;    -   2) a binding domain for the ligand; and    -   3) a transactivation domain; and

b) a DNA construct comprising:

-   -   1) the exogenous gene; and    -   2) a response element;        wherein the exogenous gene is under the control of the response        element; and binding of the DNA binding domain to the response        element in the presence of the ligand results in activation or        suppression of the gene.        A related aspect of this invention is a method for regulating        endogenous or heterologous gene expression in a transgenic        subject comprising contacting a ligand comprising a compound of        the present invention with an ecdysone receptor within the cells        of the subject wherein the cells contain a DNA binding sequence        for the ecdysone receptor and wherein formation of an ecdysone        receptor-ligand-DNA binding sequence complex induces expression        of the gene.

A fourth aspect of the present invention is a method for producing apolypeptide comprising the steps of:

a) selecting a cell which is substantially insensitive to exposure to aligand comprising a compound of the present invention;

b) introducing into the cell:

-   -   1) a DNA construct comprising:        -   i) an exogenous gene encoding the polypeptide; and        -   ii) a response element;            wherein the gene is under the control of the response            element; and    -   2) an ecdysone receptor complex comprising:        -   i) a DNA binding domain;        -   ii) a binding domain for the ligand; and        -   iii) a transactivation domain; and

c) exposing the cell to the ligand.

As well as the advantage of temporally controlling polypeptideproduction by the cell, this aspect of the invention provides a furtheradvantage, in those cases when accumulation of such a polypeptide candamage the cell, in that expression of the polypeptide may be limited toshort periods. Such control is particularly important when the exogenousgene is a therapeutic gene. Therapeutic genes may be called upon toproduce polypeptides which control needed functions, such as theproduction of insulin in diabetic patients. They may also be used toproduce damaging or even lethal proteins, such as those lethal to cancercells. Such control may also be important when the protein levelsproduced may constitute a metabolic drain on growth or reproduction,such as in transgenic plants.

Numerous genomic and cDNA nucleic acid sequences coding for a variety ofpolypeptides are well known in the art. Exogenous genetic materialuseful with the ligands of this invention include genes that encodebiologically active proteins of interest, such as, for example,secretory proteins that can be released from a cell; enzymes that canmetabolize a substrate from a toxic substance to a non-toxic substance,or from an inactive substance to an active substance; regulatoryproteins; cell surface receptors; and the like. Useful genes alsoinclude genes that encode blood clotting factors, hormones such asinsulin, parathyroid hormone, luteinizing hormone releasing factor,alpha and beta seminal inhibins, and human growth hormone; genes thatencode proteins such as enzymes, the absence of which leads to theoccurrence of an abnormal state; genes encoding cytokines or lymphokinessuch as interferons, granulocytic macrophage colony stimulating factor,colony stimulating factor-1, tumor necrosis factor, and erythropoietin;genes encoding inhibitor substances such as alpha₁-antitrypsin, genesencoding substances that function as drugs such as diphtheria andcholera toxins; and the like. Useful genes also include those useful forcancer therapies and to treat genetic disorders. Those skilled in theart have access to nucleic acid sequence information for virtually allknown genes and can either obtain the nucleic acid molecule directlyfrom a public depository, the institution that published the sequence,or employ routine methods to prepare the molecule.

For gene therapy use, the ligands described herein may be taken up inpharmaceutically acceptable carriers, such as, for example, solutions,suspensions, tablets, capsules, ointments, elixirs, and injectablecompositions. Pharmaceutical preparations may contain from 0.01% to 99%by weight of the ligand. Preparations may be either in single ormultiple dose forms. The amount of ligand in any particularpharmaceutical preparation will depend upon the effective dose, that is,the dose required to elicit the desired gene expression or suppression.

Suitable routes of administering the pharmaceutical preparations includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural) and by naso-gastric tube. It willbe understood by those skilled in the art that the preferred route ofadministration will depend upon the condition being treated and may varywith factors such as the condition of the recipient.

The ligands described herein may also be administered in conjunctionwith other pharmaceutically active compounds. It will be understood bythose skilled in the art that pharmaceutically active compounds to beused in combination with the ligands described herein will be selectedin order to avoid adverse effects on the recipient or undesirableinteractions between the compounds. Examples of other pharmaceuticallyactive compounds which may be used in combination with the ligandsinclude, for example, AIDS chemotherapeutic agents, amino acidderivatives, analgesics, anesthetics, anorectal products, antacids andantiflatulents, antibiotics, anticoagulants, antidotes, antifibrinolyticagents, antihistamines, anti-inflamatory agents, antineoplastics,antiparasitics, antiprotozoals, antipyretics, antiseptics,antispasmodics and anticholinergics, antivirals, appetite suppressants,arthritis medications, biological response modifiers, bone metabolismregulators, bowel evacuants, cardiovascular agents, central nervoussystem stimulants, cerebral metabolic enhancers, cerumenolytics,cholinesterase inhibitors, cold and cough preparations, colonystimulating factors, contraceptives, cytoprotective agents, dentalpreparations, deodorants, dermatologicals, detoxifying agents, diabetesagents, diagnostics, diarrhea medications, dopamine receptor agonists,electrolytes, enzymes and digestants, ergot preparations, fertilityagents, fiber supplements, antifungal agents, galactorrhea inhibitors,gastric acid secretion inhibitors, gastrointestinal prokinetic agents,gonadotropin inhibitors, hair growth stimulants, hematinics,hemorrheologic agents, hemostatics, histamine H₂ receptor antagonists,hormones, hyperglycemic agents, hypolipidemics, immunosuppressants,laxatives, leprostatics, leukapheresis adjuncts, lung surfactants,migraine preparations, mucolytics, muscle relaxant antagonists, musclerelaxants, narcotic antagonists, nasal sprays, nausea medicationsnucleoside analogues, nutritional supplements, osteoporosispreparations, oxytocics, parasympatholytics, parasympathomimctics,Parkinsonism drugs, Penicillin adjuvants, phospholipids, plateletinhibitors, porphyria agents, prostaglandin analogues, prostaglandins,proton pump inhibitors, pruritus medications psychotropics, quinolones,respiratory stimulants, saliva stimulants, salt substitutes, sclerosingagents, skin wound preparations, smoking cessation aids, sulfonamides,sympatholytics, thrombolytics, Tourette's syndrome agents, tremorpreparations, tuberculosis preparations, uricosuric agents, urinarytract agents, uterine contractants, uterine relaxants, vaginalpreparations, vertigo agents, vitamin D analogs, vitamins, and medicalimaging contrast media. In some cases the ligands may be useful as anadjunct to drug therapy, for example, to “turn off” a gene that producesan enzyme that metabolizes a particular drug.

For agricultural applications, in addition to the applications describedabove, the ligands of this invention may also be used to control theexpression of pesticidal proteins such as Bacillus thuringiensis (Bt)toxin. Such expression may be tissue or plant specific. In addition,particularly when control of plant pests is also needed, one or morepesticides may be combined with the ligands described herein, therebyproviding additional advantages and effectiveness, including fewer totalapplications, than if the pesticides are applied separately. Whenmixtures with pesticides are employed, the relative proportions of eachcomponent in the composition will depend upon the relative efficacy andthe desired application rate of each pesticide with respect to thecrops, pests, and/or weeds to be treated. Those skilled in the art willrecognize that mixtures of pesticides may provide advantages such as abroader spectrum of activity than one pesticide used alone. Examples ofpesticides which can be combined in compositions with the ligandsdescribed herein include fungicides, herbicides, insecticides,miticides, and microbicides.

The ligands described herein can be applied to plant foliage as aqueoussprays by methods commonly employed, such as conventional high-literhydraulic sprays, low-liter sprays, air-blast, and aerial sprays. Thedilution and rate of application will depend upon the type of equipmentemployed, the method and frequency of application desired, and theligand application rate. It may be desirable to include additionaladjuvants in the spray tank. Such adjuvants include surfactants,dispersants, spreaders, stickers, antifoam agents, emulsifiers, andother similar materials described in McCutcheon's Emulsifiers andDetergents, McCutcheon's Emulsifiers and Detergents/FunctionalMaterials, and McCutcheon's Functional Materials, all published annuallyby McCutcheon Division of MC Publishing Company (New Jersey). Theligands can also be mixed with fertilizers or fertilizing materialsbefore their application. The ligands and solid fertilizing material canalso be admixed in mixing or blending equipment, or they can beincorporated with fertilizers in granular formulations. Any relativeproportion of fertilizer can be used which is suitable for the crops andweeds to be treated. The ligands described herein will commonly comprisefrom 5% to 50% of the fertilizing composition. These compositionsprovide fertilizing materials which promote the rapid growth of desiredplants, and at the same time control gene expression.

Host Cells and Non-Human Organisms of the Invention

As described above, ligands for modulating gene expression system of thepresent invention may be used to modulate gene expression in a hostcell. Expression in transgenic host cells may be useful for theexpression of various genes of interest. The present invention providesligands for modulation of gene expression in prokaryotic and eukaryotichost cells. Expression in transgenic host cells is useful for theexpression of various polypeptides of interest including but not limitedto antigens produced in plants as vaccines, enzymes like alpha-amylase,phytase, glucanes, and xylanse, genes for resistance against insects,nematodes, fungi, bacteria, viruses, and abiotic stresses, antigens,nutraceuticals, pharmaceuticals, vitamins, genes for modifying aminoacid content, herbicide resistance, cold, drought, and heat tolerance,industrial products, oils, protein, carbohydrates, antioxidants, malesterile plants, flowers, fuels, other output traits, therapeuticpolypeptides, pathway intermediates; for the modulation of pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host; cell based assays; functionalgenomics assays, biotherapeutic protein production, proteomics assays,and the like. Additionally the gene products may be useful forconferring higher growth yields of the host or for enabling analternative growth mode to be utilized.

Thus, the present invention provides ligands for modulating geneexpression in an isolated host cell according to the invention. The hostcell may be a bacterial cell, a fungal cell, a nematode cell, an insectcell, a fish cell, a plant cell, an avian cell, an animal cell, or amammalian cell. In still another embodiment, the invention relates toligands for modulating gene expression in an host cell, wherein themethod comprises culturing the host cell as described above in culturemedium under conditions permitting expression of a polynucleotideencoding the nuclear receptor ligand binding domain comprising asubstitution mutation, and isolating the nuclear receptor ligand bindingdomain comprising a substitution mutation from the culture.

In a specific embodiment, the isolated host cell is a prokaryotic hostcell or a eukaryotic host cell. In another specific embodiment, theisolated host cell is an invertebrate host cell or a vertebrate hostcell. Preferably, the host cell is selected from the group consisting ofa bacterial cell, a fungal cell, a yeast cell, a nematode cell, aninsect cell, a fish cell, a plant cell, an avian cell, an animal cell,and a mammalian cell. More preferably, the host cell is a yeast cell, anematode cell, an insect cell, a plant cell, a zebrafish cell, a chickencell, a hamster cell, a mouse cell, a rat cell, a rabbit cell, a catcell, a dog cell, a bovine cell, a goat cell, a cow cell, a pig cell, ahorse cell, a sheep cell, a simian cell, a monkey cell, a chimpanzeecell, or a human cell. Examples of preferred host cells include, but arenot limited to, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterialspecies such as those in the genera Synechocystis, Synechococcus,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocstis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella;plant species selected from the group consisting of an apple,Arabidopsis, bajra, banana, barley, beans, beet, blackgram, chickpea,chili, cucumber, eggplant, favabean, maize, melon, millet, mungbean,oat, okra, Panicum, papaya, peanut, pea, pepper, pigeonpea, pineapple,Phaseolus, potato, pumpkin, rice, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,and wheat; animal; and mammalian host cells.

In a specific embodiment, the host cell is a yeast cell selected fromthe group consisting of a Saccharomyces, a Pichia, and a Candida hostcell.

In another specific embodiment, the host cell is a Caenorhabdus elegansnematode cell.

In another specific embodiment, the host cell is an insect cell.

In another specific embodiment, the host cell is a plant cell selectedfrom the group consisting of an apple, Arabidopsis, bajra, banana,barley, beans, beet, blackgram, chickpea, chili, cucumber, eggplant,favabean, maize, melon, millet, mungbean, oat, okra, Panicum, papaya,peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato, pumpkin,rice, sorghum, soybean, squash, sugarcane, sugarbeet, sunflower, sweetpotato, tea, tomato, tobacco, watermelon, and wheat cell.

In another specific embodiment, the host cell is a zebra fish cell.

In another specific embodiment, the host cell is a chicken cell.

In another specific embodiment, the host cell is a mammalian cellselected from the group consisting of a hamster cell, a mouse cell, arat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goatcell, a cow cell, a pig cell, a horse cell, a sheep cell, a monkey cell,a chimpanzee cell, and a human cell.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prokaryotic and eukaryotic cells are well knownin the art (see General Methods section of Examples). Cells may beharvested and the gene products isolated according to protocols specificfor the gene product.

In addition, a host cell may be chosen which modulates the expression ofthe inserted polynucleotide, or modifies and processes the polypeptideproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification [e.g., glycosylation,cleavage (e.g., of signal sequence)] of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a non-glycosylated coreprotein product. However, a polypeptide expressed in bacteria may not beproperly folded. Expression in yeast can produce a glycosylated product.Expression in eukaryotic cells can increase the likelihood of “native”glycosylation and folding of a heterologous protein. Moreover,expression in mammalian cells can provide a tool for reconstituting, orconstituting, the polypeptide's activity. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent. The present invention alsorelates to a non-human organism comprising an isolated host cellaccording to the invention. In a specific embodiment, the non-humanorganism is a prokaryotic organism or a eukaryotic organism. In anotherspecific embodiment, the non-human organism is an invertebrate organismor a vertebrate organism.

Preferably, the non-human organism is selected from the group consistingof a bacterium, a fungus, a yeast, a nematode, an insect, a fish, aplant, a bird, an animal, and a mammal. More preferably, the non-humanorganism is a yeast, a nematode, an insect, a plant, a zebrafish, achicken, a hamster, a mouse, a rat, a rabbit, a cat, a dog, a bovine, agoat, a cow, a pig, a horse, a sheep, a simian, a monkey, or achimpanzee.

In a specific embodiment, the non-human organism is a yeast selectedfrom the group consisting of Saccharomyces, Pichia, and Candida.

In another specific embodiment, the non-human organism is a Caenorhabduselegans nematode.

In another specific embodiment, the non-human organism is a plantselected from the group consisting of an apple, Arabidopsis, bajra,banana, barley, beans, beet, blackgram, chickpea, chili, cucumber,eggplant, favabean, maize, melon, millet, mungbean, oat, okra, Panicum,papaya, peanut, pea, pepper, pigeonpea, pineapple, Phaseolus, potato,pumpkin, rice, sorghum, soybean, squash, sugarcane, sugarbeet,sunflower, sweet potato, tea, tomato, tobacco, watermelon, and wheat.

In another specific embodiment, the non-human organism is a Mus musculusmouse.

Gene Expression Modulation System of the Invention

The present invention relates to a group of ligands that are useful inan ecdysone receptor-based inducible gene expression system. Aspresented herein, a novel group of ligands provides an improvedinducible gene expression system in both prokaryotic and eukaryotic hostcells. Thus, the present invention relates to ligands that are useful tomodulate expression of genes. In particular, the present inventionrelates to ligands having the ability to transactivate a gene expressionmodulation system comprising at least one gene expression cassette thatis capable of being expressed in a host cell comprising a polynucleotidethat encodes a polypeptide comprising a Group H nuclear receptor ligandbinding domain. Preferably, the Group H nuclear receptor ligand bindingis from an ecdysone receptor, a ubiquitous receptor, an orphan receptor1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptorinteracting protein-15, a liver X receptor β, a steroid hormone receptorlike protein, a liver X receptor, a liver X receptor α, a farnesoid Xreceptor, a receptor interacting protein 14, and a farnesol receptor.More preferably, the Group H nuclear receptor ligand binding domain isfrom an ecdysone receptor.

In a specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a gene expression cassette comprising a) a polynucleotide thatencodes a polypeptide comprising a transactivation domain, a DNA-bindingdomain that recognizes a response element associated with a gene whoseexpression is to be modulated; and a Group H nuclear receptor ligandbinding domain comprising a substitution mutation, and b) a secondnuclear receptor ligand binding domain selected from the groupconsisting of a vertebrate retinoid X receptor ligand binding domain, aninvertebrate retinoid X receptor ligand binding domain, an ultraspiracleprotein ligand binding domain, and a chimeric ligand binding domaincomprising two polypeptide fragments, wherein the first polypeptidefragment is from a vertebrate retinoid X receptor ligand binding domain,an invertebrate retinoid X receptor ligand binding domain, or anultraspiracle protein ligand binding domain, and the second polypeptidefragment is from a different vertebrate retinoid X receptor ligandbinding domain, invertebrate retinoid X receptor ligand binding domain,or ultraspiracle protein ligand binding domain. The gene expressionmodulation system may further comprise a second gene expression cassettecomprising: i) a response element recognized by the DNA-binding domainof the encoded polypeptide of the first gene expression cassette; ii) apromoter that is activated by the transactivation domain of the encodedpolypeptide of the first gene expression cassette; and iii) a gene whoseexpression is to be modulated.

In another specific embodiment, the gene expression modulation systemcomprises a first gene expression cassette comprising a polynucleotidethat encodes a first polypeptide comprising a DNA-binding domain thatrecognizes a response element associated with a gene whose expression isto be modulated and a nuclear receptor ligand binding domain, and asecond gene expression cassette comprising a polynucleotide that encodesa second polypeptide comprising a transactivation domain and a nuclearreceptor ligand binding domain, wherein one of the nuclear receptorligand binding domains is a Group H nuclear receptor ligand bindingdomain comprising a substitution mutation. In a preferred embodiment,the first polypeptide is substantially free of a transactivation domainand the second polypeptide is substantially free of a DNA bindingdomain. For purposes of the invention, “substantially free” means thatthe protein in question does not contain a sufficient sequence of thedomain in question to provide activation or binding activity. The geneexpression modulation system may further comprise a third geneexpression cassette comprising: i) a response element recognized by theDNA-binding domain of the first polypeptide of the first gene expressioncassette; ii) a promoter that is activated by the transactivation domainof the second polypeptide of the second gene expression cassette; andiii) a gene whose expression is to be modulated.

Wherein when only one nuclear receptor ligand binding domain is a GroupH ligand binding domain comprising a substitution mutation, the othernuclear receptor ligand binding domain may be from any other nuclearreceptor that forms a dimer with the Group H ligand binding domaincomprising the substitution mutation. For example, when the Group Hnuclear receptor ligand binding domain comprising a substitutionmutation is an ecdysone receptor ligand binding domain comprising asubstitution mutation, the other nuclear receptor ligand binding domain(“partner”) may be from an ecdysone receptor, a vertebrate retinoid Xreceptor (RXR), an invertebrate RXR, an ultraspiracle protein (USP), ora chimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from thegroup consisting of a vertebrate RXR, an invertebrate RXR, and a USP(see co-pending applications PCT/US01/09050, PCT/US02/05235, andPCT/US02/05706, incorporated herein by reference in their entirety). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

Preferably, the vertebrate RXR ligand binding domain is from a humanHomo sapiens, mouse Mus musculus, rat Ratfus norvegicus, chicken Gallusgallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Danioredio, tunicate Polyandrocarpa misakiensis, or jellyfish Tripedaliacysophora RXR.

Preferably, the invertebrate RXR ligand binding domain is from a locustLocusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodid tickAmblyomma americanum RXR homolog 1 (“AmaRXR1”), a ixodid tick Amblyommaamericanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilatorRXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TrRXR”),a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicaeRXR homolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.

Preferably, the chimeric RXR ligand binding domain comprises at leasttwo polypeptide fragments selected from the group consisting of avertebrate species RXR polypeptide fragment, an invertebrate species RXRpolypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebratespecies RXR homolog polypeptide fragment. A chimeric RXR ligand bindingdomain for use in the present invention may comprise at least twodifferent species RXR polypeptide fragments, or when the species is thesame, the two or more polypeptide fragments may be from two or moredifferent isoforms of the species RXR polypeptide fragment.

In a preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone invertebrate species RXR polypeptide fragment.

In a more preferred embodiment, the chimeric RXR ligand binding domaincomprises at least one vertebrate species RXR polypeptide fragment andone non-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

In a specific embodiment, the gene whose expression is to be modulatedis a homologous gene with respect to the host cell. In another specificembodiment, the gene whose expression is to be modulated is aheterologous gene with respect to the host cell.

The ligands for use in the present invention as described below, whencombined with the ligand binding domain of the nuclear receptor(s),which in turn are bound to the response element linked to a gene,provide the means for external temporal regulation of expression of thegene. The binding mechanism or the order in which the various componentsof this invention bind to each other, that is, for example, ligand toligand binding domain, DNA-binding domain to response element,transactivation domain to promoter, etc., is not critical.

In a specific example, binding of the ligand to the ligand bindingdomain of a Group H nuclear receptor and its nuclear receptor ligandbinding domain partner enables expression or suppression of the gene.This mechanism does not exclude the potential for ligand binding to theGroup H nuclear receptor (GHNR) or its partner, and the resultingformation of active homodimer complexes (e.g. GHNR+GHNR orpartner+partner). Preferably, one or more of the receptor domains isvaried producing a hybrid gene switch. Typically, one or more of thethree domains, DBD, LBD, and transactivation domain, may be chosen froma source different than the source of the other domains so that thehybrid genes and the resulting hybrid proteins are optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL4 protein from yeast (see Sadowski, et al. (1988) Nature, 335:563-564) or LexA protein from Escherichia coli (see Brent and Ptashne(1985), Cell, 43: 729-736), or synthetic response elements specific fortargeted interactions with proteins designed, modified, and selected forsuch specific interactions (see, for example, Kim, et al. (1997), Proc.Natl. Acad. Sci., USA, 94: 3616-3620) to accommodate hybrid receptors.Another advantage of two-hybrid systems is that they allow choice of apromoter used to drive the gene expression according to a desired endresult. Such double control can be particularly important in areas ofgene therapy, especially when cytotoxic proteins are produced, becauseboth the timing of expression as well as the cells wherein expressionoccurs can be controlled. When genes, operably linked to a suitablepromoter, are introduced into the cells of the subject, expression ofthe exogenous genes is controlled by the presence of the system of thisinvention. Promoters may be constitutively or inducibly regulated or maybe tissue-specific (that is, expressed only in a particular type ofcells) or specific to certain developmental stages of the organism.

The ecdysone receptor is a member of the nuclear receptor superfamilyand classified into subfamily 1, group H (referred to herein as “Group Hnuclear receptors”). The members of each group share 40-60% amino acididentity in the E (ligand binding) domain (Laudet et al., A UnifiedNomenclature System for the Nuclear Receptor Subfamily, 1999; Cell 97:161-163). In addition to the ecdysone receptor, other members of thisnuclear receptor subfamily 1, group H include: ubiquitous receptor (UR),orphan receptor 1 (OR-1), steroid hormone nuclear receptor 1 (NER-1),retinoid X receptor interacting protein-15 (RIP-15), liver X receptor β(LXRβ), steroid hormone receptor like protein (RLD-1), liver X receptor(LXR), liver X receptor α (LXRα), farnesoid X receptor (FXR), receptorinteracting protein 14 (RIP-14), and farnesol receptor (HRR-1

In particular, described herein are novel ligands useful in a geneexpression modulation system comprising a Group H nuclear receptorligand binding domain comprising a substitution mutation. This geneexpression system may be a “single switch”-based gene expression systemin which the transactivation domain, DNA-binding domain and ligandbinding domain are on one encoded polypeptide. Alternatively, the geneexpression modulation system may be a “dual switch”- or“two-hybrid”-based gene expression modulation system in which thetransactivation domain and DNA-binding domain are located on twodifferent encoded polypeptides.

An ecdysone receptor-based gene expression modulation system of thepresent invention may be either heterodimeric or homodimeric. Afunctional EcR complex generally refers to a heterodimeric proteincomplex consisting of two members of the steroid receptor family, anecdysone receptor protein obtained from various insects, and anultraspiracle (USP) protein or the vertebrate homolog of USP, retinoid Xreceptor protein (see Yao, et al. (1993) Nature 366, 476-479; Yao, etal., (1992) Cell 71, 63-72). However, the complex may also be ahomodimer as detailed below. The functional ecdysteroid receptor complexmay also include additional protein(s) such as immunophilins. Additionalmembers of the steroid receptor family of proteins, known astranscriptional factors (such as DHR38 or betaFTZ-1), may also be liganddependent or independent partners for EcR, USP, and/or REX.Additionally, other cofactors may be required such as proteins generallyknown as coactivators (also termed adapters or mediators). Theseproteins do not bind sequence-specifically to DNA and are not involvedin basal transcription. They may exert their effect on transcriptionactivation through various mechanisms, including stimulation ofDNA-binding of activators, by affecting chromatin structure, or bymediating activator-initiation complex interactions. Examples of suchcoactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1,TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuouscoactivator C response element B binding protein, CBP/p300 (for reviewsee Glass et al., Curr. Opin. Cell Biol. 9:222-232, 1997). Also, proteincofactors generally known as corepressors (also known as repressors,silencers, or silencing mediators) may be required to effectivelyinhibit transcriptional activation in the absence of ligand. Thesecorepressors may interact with the unliganded ecdysone receptor tosilence the activity at the response element. Current evidence suggeststhat the binding of ligand changes the conformation of the receptor,which results in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N-CoR and SMRT (for review, see Horwitzet al. Mol. Endocrinol. 10: 1167-1177, 1996). These cofactors may eitherbe endogenous within the tell or organism, or may be added exogenouslyas transgenes to be expressed in either a regulated or unregulatedfashion. Homodimer complexes of the ecdysone receptor protein, USP, orRXR may also be functional under some circumstances.

The ecdysone receptor complex typically includes proteins that aremembers of the nuclear receptor superfamily wherein all members aregenerally characterized by the presence of an amino-terminaltransactivation domain, a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”) separated from the DBD by a hinge region. As usedherein, the term “DNA binding domain” comprises a minimal polypeptidesequence of a DNA binding protein, up to the entire length of a DNAbinding protein, so long as the DNA binding domain functions toassociate with a particular response element. Members of the nuclearreceptor superfamily are also characterized by the presence of four orfive domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889-895 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for ecdysone response elements. These domains may beeither native, modified, or chimeras of different domains ofheterologous receptor proteins. The EcR receptor, like a subset of thesteroid receptor family, also possesses less well-defined regionsresponsible for heterodimerization properties. Because the domains ofnuclear receptors are modular in nature, the LBD, DBD, andtransactivation domains may be interchanged.

Gene switch systems are known that incorporate components from theecdysone receptor complex. However, in these known systems, whenever EcRis used it is associated with native or modified DNA binding domains andtransactivation domains on the same molecule. USP or RXR are typicallyused as silent partners. It has previously been shown that when DNAbinding domains and transactivation domains are on the same molecule thebackground activity in the absence of ligand is high and that suchactivity is dramatically reduced when DNA binding domains andtransactivation domains are on different molecules, that is, on each oftwo partners of a heterodimeric or homodimeric complex (seePCT/US01/09050).

Method of Modulating Gene Expression of the Invention

The present invention also relates to methods of modulating geneexpression in a host cell using a gene expression modulation systemaccording to the invention. Specifically, the present invention providesa method of modulating the expression of a gene in a host cellcomprising the steps of: a) introducing into the host cell a geneexpression modulation system according to the invention; and b)introducing into the host cell a ligand; wherein the gene to bemodulated is a component of a gene expression cassette comprising: i) aresponse element comprising a domain recognized by the DNA bindingdomain of the gene expression system; ii) a promoter that is activatedby the transactivation domain of the gene expression system; and iii) agene whose expression is to be modulated, whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

The invention also provides a method of modulating the expression of agene in a host cell comprising the steps of: a) introducing into thehost cell a gene expression modulation system according to theinvention; b) introducing into the host cell a gene expression cassetteaccording to the invention, wherein the gene expression cassettecomprises i) a response element comprising a domain recognized by theDNA binding domain from the gene expression system; ii) a promoter thatis activated by the transactivation domain of the gene expressionsystem; and iii) a gene whose expression is to be modulated; and c)introducing into the host cell a ligand; whereby upon introduction ofthe ligand into the host cell, expression of the gene is modulated.

The present invention also provides a method of modulating theexpression of a gene in a host cell comprising a gene expressioncassette comprising a response element comprising a domain to which theDNA binding domain from the first hybrid polypeptide of the geneexpression modulation system binds; a promoter that is activated by thetransactivation domain of the second hybrid polypeptide of the geneexpression modulation system; and a gene whose expression is to bemodulated; wherein the method comprises the steps of: a) introducinginto the host cell a gene expression modulation system according to theinvention; and b) introducing into the host cell a ligand; whereby uponintroduction of the ligand into the host, expression of the gene ismodulated.

Genes of interest for expression in a host cell using methods disclosedherein may be endogenous genes or heterologous genes. Nucleic acid oramino acid sequence information for a desired gene or protein can belocated in one of many public access databases, for example, GENBANK,EMBL, Swiss-Prot, and PIR, or in many biology related journalpublications. Thus, those skilled in the art have access to nucleic acidsequence information for virtually all known genes. Such information canthen be used to construct the desired constructs for the insertion ofthe gene of interest within the gene expression cassettes used in themethods described herein.

Examples of genes of interest for expression in a host cell usingmethods set forth herein include, but are not limited to: antigensproduced in plants as vaccines, enzymes like alpha-amylase, phytase,glucanes, and xylanse, genes for resistance against insects, nematodes,fungi, bacteria, viruses, and abiotic stresses, nutraceuticals,pharmaceuticals, vitamins, genes for modifying amino acid content,herbicide resistance, cold, drought, and heat tolerance, industrialproducts, oils, protein, carbohydrates, antioxidants, male sterileplants, flowers, fuels, other output traits, genes encodingtherapeutically desirable polypeptides or products that may be used totreat a condition, a disease, a disorder, a dysfunction, a geneticdefect, such as monoclonal antibodies, enzymes, proteases, cytokines,interferons, insulin, erythropoietin, clotting factors, other bloodfactors or components, viral vectors for gene therapy, virus forvaccines, targets for drug discovery, functional genomics, andproteomics analyses and applications, and the like.

Measuring Gene Expression/Transcription

One useful measurement of the methods of the invention is that of thetranscriptional state of the cell including the identities andabundances of RNA, preferably mRNA species. Such measurements areconveniently conducted by measuring cDNA abundances by any of severalexisting gene expression technologies.

Nucleic acid array technology is a useful technique for determiningdifferential mRNA expression. Such technology includes, for example,oligonucleotide chips and DNA microarrays. These techniques rely on DNAfragments or oligonucleotides which correspond to different genes orcDNAs which are immobilized on a solid support and hybridized to probesprepared from total mRNA pools extracted from cells, tissues, or wholeorganisms and converted to cDNA. Oligonucleotide chips are arrays ofoligonucleotides synthesized on a substrate using photolithographictechniques. Chips have been produced which can analyze for up to 1700genes. DNA microarrays are arrays of DNA samples, typically PCRproducts, that are robotically printed onto a microscope slide. Eachgene is analyzed by a full or partial-length target DNA sequence.Microarrays with up to 10,000 genes are now routinely preparedcommercially. The primary difference between these two techniques isthat oligonucleotide chips typically utilize 25-mer oligonucleotideswhich allow fractionation of short DNA molecules whereas the larger DNAtargets of microarrays, approximately 1000 base pairs, may provide moresensitivity in fractionating complex DNA mixtures.

Another useful measurement of the methods of the invention is that ofdetermining the translation state of the cell by measuring theabundances of the constituent protein species present in the cell usingprocesses well known in the art.

Where identification of genes associated with various physiologicalfunctions is desired, an assay may be employed in which changes in suchfunctions as cell growth, apoptosis, senescence, differentiation,adhesion, binding to a specific molecules, binding to another cell,cellular organization, organogenesis, intracellular transport, transportfacilitation, energy conversion, metabolism, myogenesis, neurogenesis,and/or hematopoiesis is measured.

In addition, selectable marker or reporter gene expression may be usedto measure gene expression modulation using the present invention

Other methods to detect the products of gene expression are well knownin the art and include Southern blots (DNA detection), dot or slot blots(DNA, RNA), northern blots (RNA), RT-PCR (RNA), western blots(polypeptide detection), and ELISA (polypeptide) analyses. Although lesspreferred, labeled proteins can be used to detect a particular nucleicacid sequence to which it hybridizes.

In some cases it is necessary to amplify the amount of a nucleic acidsequence. This may be carried out using one or more of a number ofsuitable methods including, for example, polymerase chain reaction(“PCR”), ligase chain reaction (“LCR”), strand displacementamplification (“SDA”), transcription-based amplification, and the like.PCR is carried out in accordance with known techniques in which, forexample, a nucleic acid sample is treated in the presence of a heatstable DNA polymerase, under hybridizing conditions, with one pair ofoligonucleotide primers, with one primer hybridizing to one strand(template) of the specific sequence to be detected. The primers aresufficiently complementary to each template strand of the specificsequence to hybridize therewith. An extension product of each primer issynthesized and is complementary to the nucleic acid template strand towhich it hybridized. The extension product synthesized from each primercan also serve as a template for further synthesis of extension productsusing the same primers. Following a sufficient number of rounds ofsynthesis of extension products, the sample may be analyzed as describedabove to assess whether the sequence or sequences to be detected arepresent.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention.

EXAMPLES General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) andby T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology. A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, M A (1989). All reagents, restriction enzymes and materialsused for the growth and maintenance of host cells were obtained fromAldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit,Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St.Louis, Mo.) unless otherwise specified.

Manipulations of genetic sequences may be accomplished using the suiteof programs available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).Where the GCG program “Pileup” is used the gap creation default value of12, and the gap extension default value of 4 may be used. Where the CGC“Gap” or “Bestfit” program is used the default gap creation penalty of50 and the default gap extension penalty of 3 may be used. In any casewhere GCG program parameters are not prompted for, in these or any otherGCG program, default values may be used.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimoles, “μg” means microgram(s), “mg” means milligram(s), “A” meansadenine or adenosine, “T” means thymine or thymidine, “G” means guanineor guanosine, “C” means cytidine or cytosine, “×g” means times gravity,“nt” means nucleotide(s), “aa” means amino acid(s), “bp” means basepair(s), “kb” means kilobase(s), “k” means kilo, “μ” means micro, “° C.”means degrees Celsius, “C” in the context of a chemical equation meansCelsius, “THF” means tetrahydrofuran, “DME” means dimethoxyethane, “DMF”means dimethylformamide, “NMR” means nuclear magnetic resonance, “psi”refers to pounds per square inch, and “TLC” means thin layerchromatography.

Example 1 Preparation of Compounds

The compounds of the present invention may be made according to thefollowing synthesis routes.

1.1 Preparation of Isopropylidene-hydrazine

A 500 mL 3-neck flask was fitted with a mechanical stirrer, thermometer,and addition funnel. The vessel was charged with 140 mL of methanol, andthen cooled to −5° C. in an ice/salt bath, BaO (7.5 g, 50 mmole) wasthen added portion-wise over 5 minutes. Gas evolution and an exothermwere observed. A maximum temperature of 5° C. was reached. The reactionwas cooled down to 0° C., and hydrazine monohydrate (32 g, 0.64 moles)was added in one portion, causing the mixture to warm to 5° C. Thevessel was cooled down to 0° C. and the mixture was stirred for 10minutes. A solution of acetone (37 g, 0.64 moles) in 40 mL of methanolwas added drop-wise over 1 hour at 5° C. Stirring was continued,allowing the reaction to warm slowly to room temperature. Stirring wascontinued overnight, but if the reaction was allowed to proceed for onlyone hour, quite satisfactory yields were obtained. ¹H NMR indicated theabsence of acetone and a complete reaction. Ether (300 mL) was added,which caused more solid to precipitate. Celite 545 and MgSO₄ were added,the mixture was filtered through a bed of Celite on S&S sharkskin paperin a sintered glass funnel, and most of the ether was removed at orbelow room temperature on a rotary evaporator. The solution was thengently distilled. The distillate was placed in a clean flask and theremaining methanol was removed by rotovap evaporation withoutapplication of heat. The process was monitored by ¹H NMR until <2.5%methanol remained. Isopropylidene-hydrazine was obtained as a slightlycloudy colorless liquid (41 g, 89%), and used as such in subsequentreactions. ¹H NMR (300 MHz, CDCl3) δ(ppm): 4.85 (br, 2H), 1.93 (s, 3H),1.77 (s, 3H).

1.2 Preparation of sec-butylidene-hydrazine

A 1 L flask equipped with a mechanical stirrer, thermometer, andaddition funnel was charged with 250 mL of methanol and cooled to 0° C.Barium oxide (15.3 g, 100 mmol) was added portion-wise with exotherm.The reaction was cooled to 0° C., and hydrazine monohydrate (64.1 g,1.28 mol) was slowly added. The reaction mixture was stirred for 10minutes, after which methylethylketone (92.3 g, 1.28 mol) was addeddrop-wise over a 30 minute period. The reaction was then stirred for 1hour, maintaining a temperature below 8° C. ¹H NMR indicated the absenceof ketone and a complete reaction. Ether (200 mL) was then added, andthe BaO was filtered through a bed of silica gel. The resultant clearfiltrate was liberated of methanol on a rotary evaporator at or belowroom temperature, the receptacle was changed, and the product hydrazonewas distilled using a water bath set at 38° C. After discarding a smallforerun and residue, 70 g of sec-butylidene-hydrazine were obtained as aclear colorless liquid. The material was stored under refrigeration. ¹HNMR (300 MHz, CDCl3) δ (ppm): 4.9 (br, 2H), 2.25 (q, 2H), 1.78 (s, 3H),1.07 (t, 3H). Angew. Chemie, 94, 2, 133 (1982).

1.3 Preparation of cyclopentylidene-hydrazine

Cyclopentylidene-hydrazine was prepared in an analogous manner in 61%yield. The material was stored under refrigeration. ¹H NMR (300 MHz,CDCl3) δ (ppm): 4.9 (br, 2H), 2.35 (t, 2H), 2.2 (t, 2H), 1.9 (m, 2H),1.78 (m, 3H), 1.07 (t, 3H).

1.4 Preparation of (tetrahydro-pyran-4-ylidene)-hydrazine

Tetrahydro-4H-pyran-4-one (10 g, 0.1 mol) and methanol (150 mL) werecharged to a 500 mL three-necked flask fitted with an addition funnel.The vessel was cooled in an ice bath and kept under a nitrogenatmosphere while a solution of hydrazine (2.5 g, 50 mol) in 50 mL ofmethanol was added drop-wise over a period of one hour. A whiteprecipitate formed after about half of the hydrazine was added. Stirringwas continued overnight, and subsequent TLC indicated one product. Themethanol was removed in vacuo, and the residue was slurried in hexaneand filtered to yield 9.1 g (46.4 mol)N,N′-bis-(tetrahydro-pyran-4-ylidene)-hydrazine as a white solid. ¹H NMR(300 MHz, CDCl3) δ (ppm): 3.88 (t, 2H), 3.77 (t, 2H), 2.67 (t, 2H), 2.50(t, 2H).

This material was charged along with hydrazine (1.6 g, 50 mol) to a 300mL flask fitted with a magnetic stirrer and reflux condensor containing100 mL of absolute ethanol, which had been freshly dried by azeotropicdistillation with the aid of a Dean-Stark trap. The reaction mixture washeated at reflux for 6 hours at which time ¹H NMR analysis indicatedonly a trace of azine. The reaction mixture was cooled and the solventremoved on a rotary evaporator at 35° C. (Higher temperatures acceleratereversion to the azine). The remaining ethanol was eliminated byrepeated addition of carbon tetrachloride, followed by removal in vacuo.Tetrahydro-pyran-4-ylidene-hydrazine containing about 5-10% azine couldbe isolated in this manner. Upon standing at room temperature, thehydrazone disproportionates to the azine in a matter of days. ¹H NMR(300 MHz, CDCl3) δ (ppm): 5.0 (br, 2H), 3.80 (m, 4H), 2.39 (m, 4H).

1.5 Preparation of Indan-2-ylidene-hydrazine

Indan-2-one (25 g) and methanol (25 mL) were charged to a 500 mL 3-neckflask with an addition funnel and magnetic stirrer under an atmosphereof nitrogen. A solution of hydrazine monohydrate (4.73 g) in 50 mL ofmethanol was added drop-wise over one hour at room temperature. Stirringwas continued overnight. The resultant precipitate (ca. 15.5 g) wascollected, and shaken with a water/chloroform mixture. The chloroformlayer was dried, and the solvent was removed in vacuo to give areddish-white solid. The original supernatant contained more of theintended N,N′-di-indan-2-ylidene-hydrazine. ¹H NMR (300 MHz, CDCl3) δ(ppm); 7.4 (m, 1H), 7.3 (m, 3H), 3.93 (s, 2H), 3.82 (s, 2H).

The intermediate azine (15.35 g) was dissolved in 200 mL of absoluteethanol in a 500 mL round bottom flask. Approximately 50 mL of thesolvent was distilled off, after which a solution of 1.9 g anhydroushydrazine in ethanol was added. The reaction was heated at reflux for 90minutes, at which time the starting azine compound could no longer bedetected by ¹H NMR. Indan-2-ylidene-hydrazine was isolated as a brownsolid. ¹H NMR (300 MHz, CDCl3) δ (ppm); 7.3 (m, 4H), 5.05 (br s, 2H),3.76 (s, 2H), 3.58 (s, 2H). The purity of commercial indan-2-one may beparticularly critical for the success of this reaction.

1.6 Preparation of (1-methyl-piperidin-4-ylidene)-hydrazine

1-Methyl-piperidin-4-one (22.6 g, 0.2 mol) and methanol (200 mL) werecharged to a 500 mL three-necked flask fitted with an addition funnel.The vessel was cooled in an ice bath and kept under a nitrogenatmosphere while a solution of hydrazine monohydrate (5 g, 0.1 mol) in50 mL of methanol was added drop-wise over a period of one hour. A whiteprecipitate formed after about half of the hydrazine was added. Stirringwas continued overnight, and subsequent TLC indicated one product. Themethanol was removed in vacuo, and the residue was slurried in hexaneand filtered to provideN,N′-bis-(1-methyl-piperidin-4-ylidene)-hydrazine. ¹H NMR (300 MHz,CDCl3) δ (ppm): 2.6 (m, 8H), 2.5 (m, 8H), 2.33 (s, 6H).

This material was charged along with anhydrous hydrazine (5.2 g, 0.162mol) to a 500 mL flask fitted with a magnetic stirrer and refluxcondenser containing 200 mL of absolute ethanol, which had been freshlydried by azeotropic distillation with the aid of a Dean-Stark trap. Theclear yellow reaction mixture was heated at reflux for 4 hours at whichtime ¹H NMR analysis indicated a small amount of azine. An additional 2g of hydrazine was added and heating was continued for an additional 4hours. The reaction mixture was cooled and the solvent was removed on arotary evaporator. (Higher temperatures accelerate reversion to theazine). The remaining ethanol was eliminated by repeated addition ofcarbon tetrachloride followed by removal in vacuo.(1-Methyl-piperidin-4-ylidene)-hydrazine was obtained as a yellow oil ina 12.5 g quantity, containing about 5% azine. ¹H NMR (300 MHz, CDCl3) δ(ppm): 5.0 (br, 2H), 2.5 (m, 4H), 2.4 (m, 4H), 2.33 (s, 6H).

1.7 Preparation of Benzylidene-hydrazine

A 1 L flask equipped with a mechanical stirrer, thermometer, andaddition funnel was charged with 250 mL of methanol and cooled to 0° C.Barium oxide (9.8 g, 64 mmol) was added portion-wise with exotherm. Thereaction was cooled to 0° C., and hydrazine monohydrate (64.1 g, 1.28mol) was slowly added. The reaction mixture was stirred for 10 minutes,after which time benzaldehyde (135.8 g, 1.28 mol) was added drop-wiseover a 30 minute period. The reaction was then stirred for 1 hour, whilemaintaining a temperature below 8 (C. ¹H NMR indicated the absence ofketone and a complete reaction. Ether (200 mL) was added, and the BaOwas filtered through a bed of silica gel. The resultant clear filtratewas liberated of methanol on a rotary evaporator at or below roomtemperature. A large mass of bright yellow solid azine (¹H-NMR (300 MHz,CDCl3) δ (ppm): 8.67 (s, 2H), 7.85 (m, 4H), 7.47 (m, 6H)), was removedby filtration, and the filtrate was distilled to provide ca. 12 g ofdistillate benzylidene-hydrazine. The material was stored underrefrigeration. ¹H-NMR (300 MHz, CDCl3) δ (ppm): 7.74 (s, 1H), 7.53 (m,2H), 7.35 (m, 3H), 5.5 (br, 2H).

1.8 Preparation of (1-Phenyl-ethylidene)-hydrazine

A 1 L flask equipped with a mechanical stirrer, thermometer, andaddition funnel was charged with 300 mL of methanol and cooled to 0° C.Barium oxide (8.4 g, 55 mmol) was added portion-wise with exotherm. Thereaction was cooled again to 0° C., and hydrazine monohydrate (54.6 g,1.09 mol) was slowly added. The reaction mixture was stirred for 10minutes, after which benzaldehyde (131.5 g, 1.09 mol) was addeddrop-wise over a 30 minute period. The reaction was then stirred for 1hour, while maintaining a temperature below 8° C. ¹H NMR indicated theabsence of ketone and a complete reaction. Ether (200 mL) was added, andthe BaO was filtered through a bed of silica gel. The resultant clearfiltrate was liberated of methanol on a rotary evaporator at or belowroom temperature. The remaining concentrate was distilled at ca. 1 torrand the intended (1-phenyl-ethylidene)-hydrazine was collected at 91-94°C. in a 50 g quantity. ¹H-NMR (300 MHz, CDCl3) δ (ppm): 7.65 (m, 2H),7.35 (m, 3H), 5.37 (br, 2H), 2.13 (s, 3H). During the distillation, abright yellow solid of N,N′-bis-(1-phenyl-ethylidene)-hydrazine appearedin the distillation vessel. ¹H-NMR (300 MHz, CDCl3) δ (ppm): 7.95 (m,4H), 7.45 (m, 6H), 2.32 (s, 6H).

1.9 Preparation of (2,2,2-Trifluoro-1-methylethylidene)-hydrazine

Hydrazine monohydrate (69 g, 1.37 mol) was charged to a 300 mLsingle-neck flask fitted with a magnetic stirrer and addition funnel.The vessel was cooled in an ice bath, as trifluoroacetone (77 g, 0.687moles) was added drop-wise over a period of hours. The reaction mixturewas stirred for an additional hour and extracted several times withether. The solvent was removed from the combined ether extracts,providing about 50 g of a waxy solid. This material was distilled atatmospheric pressure, yielding ca. 13 g of(2,2,2-trifluoro-1-methyl-ethylidene)-hydrazine as a clear colorlessdistillate. ¹H-NMR (300 MHz, CDCl3) δ (ppm): 5.7 (br, 2H), 1.87 (s, 3H);b.p. 135° C.

1.10 Preparation of 3,5-Dimethyl-benzaldehyde oxime

A 2 L round bottom flask was set up with a mechanical stirrer,thermometer, N₂ inlet and reflux condensor. A solution of3,5-dimethyl-benzaldehyde (70 g. 522 mmol) in 300 mL of methanol wasadded, followed by the addition of sodium acetate (44 g, 536 mmol).Hydroxylamine-HCl (37 g, 532 mmol) was added portion-wise over 5minutes, during which time the maximum temperature reached withoutcooling was 27° C. The mixture was stirred for an additional 2 hours atroom temperature. TLC (10% ethyl acetate in hexane) indicated theabsence of the starting aldehyde and the appearance of oxime. Most ofthe methanol was removed on a rotary evaporator, resulting in theformation of a precipitate. Ether and water were added to theconcentrated suspension, and then the ether layer was collected, driedover MgSO₄, and removed on a rotary evaporator. The white crystallinematerial was air-dried, resulting in 77 g (100%) of3,5-dimethyl-benzaldehyde oxime. ¹H-NMR (300 MHz, CDCl3) δ (ppm): 8.09(s, 1H), 7.22 (s, 2H), 7.05 (s, 1H), 2.33 (s, 6H).

1.11 Preparation of Benzohydroximoyl Chloride Method A:

A 125 mL round bottom flask was fitted with a magnetic stirrer,thermometer, and pressure-equalized addition funnel. Benzaldehyde oxime(25 g, 0.206 mole) dissolved in 40 mL of CCl₄ was charged to the vessel.The reaction was protected from bright light as a solution of t-butylhypochlorite (12.1 g, prepared by the action of NaOCl on t-butyl alcoholaccording to Organic Syntheses, Volume 5, p. 183) in 20 mL of CCl₄ wasadded drop-wise over a period of 40 minutes. A transient exotherm andaqua color developed. Stirring was continued overnight at roomtemperature, after which time most of the CCl₄ from the yellow reactionmixture was removed in vacuo. Pentane was added and the solution waschilled, causing crystal formation of the intended hydroximoyl chlorideat a yield of 9.0 g. ¹H-NMR (200 MHz, CDCl3) δ (ppm): 8.4 (s, 1H, N—OH),7.87 (m, 2H), 7.45 (m, 3H).

Method B:

A 100 mL 3-neck round bottom flask, equipped with a magnetic stirrer andthermometer, was charged with 40 mL of dichloroethane, 1.06 g ofbenzaldehyde oxime, and 10 mL of isopropanol. The vessel was cooled to−12° C. in an ice salt bath. T-butyl hypochlorite was added (optionallyas a solution in dichloroethane) drop-wise over several minutes withrapid stirring, while maintaining the temperature below 10° C. A flashof blue color was observed for several seconds. The mixture was stirredfor 15 minutes with continued chilling. The solvent and by-productt-butyl alcohol were removed on a rotary evaporator and chased severaltimes with chloroform. After the third chase, a powder formed, which waswashed again with chloroform, resulting in crystal formation of 1.32 gof the product. TLC indicated a single spot which eluted slightly higherthan the starting oxime, Rf=0.4; Rf(oxime)=0.32 (4:1 hexane:ethylacetate). The product benzohydroximoyl chloride was stored in thefreezer. ¹H-NMR (300 MHz, CDCl3) δ (ppm): 8.63 (s, 1H, N—OH), 7.87 (m,2H), 7.45 (m, 3H).

Caution; T-butylhypochlorite is odoriforous and a severe lachyrmator.The benzohydroximoyl chloride may not be thermally stable, thereforehandle with a non-metal spatula, protect from strong light, and store inthe freezer. McGillivray, G.; ten Krooden, E.; S. Africa J. Chem. 986,39(1).

The following additional hydroximoyl chlorides were prepared by MethodB:

3,5-dimethylbenzohydroximoyl chloride: a white solid aftercrystallization from cold pentane, pentane/CHCl₃ or heptane. ¹H-NMR (300MHz, CDCl₃) δ (ppm): 8.4 (br, 1H), 7.45 (s, 2H), 7.12 (s, 1H), 2.35 (s,6H).

4-Phenylbenzohydroximoyl chloride: 87% yield. ¹H-NMR (300 MHz, CDCl₃) δ(ppm): 7.95 (d, 2H), 7.65 (m, 4H), 7.37-7.5 (m, 3H), 1.65 (br s, 1H).

4-Chlorobenzohydroximoyl chloride: flocculent solid, 100% yield. ¹H-NMR(300 MHz, CDCl₃) δ (ppm): 7.8 (d, 2H), 7.4 (d, 2H), 1.7 (br s, 1H). Rf(1:1 hexane:ethyl acetate)=0.63.

4-Trifluoromethoxybenzohydroximoyl chloride: 98% yield. ¹H-NMR (300 MHz,CDCl₃) δ (ppm): 7.95 (s, 1H, N—OH), 7.3 (d, 2H).

3-Trifluoromethylbenzohydroximoyl chloride: 99% yield. ¹H-NMR (300 MHz,CDCl₃) δ (ppm): 8.12 (s, 1H), 8.11 (s, 1H), 8.08 (d, 1H), 7.72 (d, 1H),7.55 (t, 1H).

2-Methoxybenzohydroximoyl chloride: 99% yield. ¹H-NMR (300 MHz, CDCl₃) δ(ppm): 9.6 (s, 1H), 7.6 (d, 1H), 7.34 (t, 1H), 7.0 (m, 2H), 3.9 (s, 3H).Rf (1:1 hexane:ethyl acetate)=0.5.

2,3-[1,3]Dioxole-benzohydroximoyl chloride. ¹H-NMR (300 MHz, CDCl₃) δ(ppm): 7.95 (br s, 1H, N—OH), 7.4 (d, 1H), 7.3 (s, 1H), 6.85 (d, 1),6.05 (s, 2H).

2,4-Dimethoxybenzohydroximoyl chloride. ¹H-NMR (300 MHz, CDCl₃) δ (ppm):7.55 (d, 1H), 6.5 (d, 1H), 6.45 (s, 1H), 3.96 (s, 3H).

3-Nitrobenzohydroximoyl chloride: 100% yield. ¹H-NMR (300 MHz, CDCl₃) δ(ppm): 8.71 (s, 1H, N—OH), 8.45 (s, 1H), 8.30 (d, 1H), 8.19 (d, 1H),7.65 (t, 1H). Rf (1:1 hexane:ethyl acetate)=0.5.

1.12 Preparation of aryl-[1,2]oxadiazol-4-ylamines Method A-1

A solution of isopropylidene-hydrazine (0.59 g, 8 mmol) in 15 mL ofchloroform and an aqueous solution of K₂CO₃ (0.5 g in 3 mL of water)were mixed and cooled in a 50 mL round-bottom flask chilled with icewater. A solution of benzohydroximoyl chloride (0.51 g, 3.2 mmol) in 10mL of chloroform was added slowly with vigorous magnetic stirring. Theice batch was replaced with a 40° C. water bath and the mixture wasstirred at 40° C. for 2 hours and then monitored by TLC (1:1 ethylacetate:hexanes). When progression of the reaction began tosignificantly decelerate, the mixture was worked up by the addition of10 mL of water and 60 mL of chloroform or methylene chloride. Theorganic layer was removed in a separatory funnel, dried over MgSO₄, andthe solvent removed on a rotary evaporator to yield a semi-solid. Thecrude product was triturated with 2% ether in hexanes (30 mL), bymagnetic stirring in a round bottom flask or manipulating the materialwith a spatula. Filtration and air-drying provided5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-ylamine in ca. 40% yield.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.77 (m, 2H), 7.48 (m, 3H), 3.4 (br),1.55 (s, 6H).

The following additional oxadiazolines were prepared by method A-1:

3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine: 52% yield,trituration from pentane. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.72 (d, 2H),7.42 (d, 2H), 3.5 (s, 1H), 1.6 (s, 1H), 1.54 (s, 6H). Rf=0.46 (1:1 ethylacetate:hexane).

3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine: 35%yield, trituration from 10% ether in hexane or ethyl acetatehexanegradient silica gel chromatography. ¹H-NMR (300 MHz, CDCl₃) δ (ppm);7.22 (s, 1H), 7.87 (s, 1H), 7.85 (s, 1H), 6.0 (s, 2H), 3.5 (br s, 2H),1.52 (s, 6H).

3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine. ¹H-NMR(300 MHz, CDCl₃) δ (ppm): 7.5 (d, 2H), 7.05 (m, 2H), 3.91 (s, 3H), 3.7(br s, 2H), 1.54 (s, 6H). Ethyl acetate/hexane gradient silica gelchromatography, Rf=−0.25 (1:1 ethyl acetate:hexane).

3-(3-Trifluoromethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine:trituration from heptane, 47% yield. ¹H-NMR (300 MHz, CDCl₃) δ (ppm):8.1 (s, 1H), 7.95 (d, 1H), 7.7 (d, 1H), 7.55 (t, 1H), 3.5 (br s, 2H),1.57 (s, 6H).

3-(4-Trifluoromethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine:trituration from heptane, 28% yield. ¹H-NMR (300 MHz, CDCl₃) δ (ppm):7.85 (d, 2H), 7.3 (d, 2H), 3.55 (br s, 2H), 1.55 (s, 6H). Ethylacetate/hexane gradient silica gel chromatography.

3-Biphenyl-4-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine: triturationfrom pentane, 49% yield. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.85 (d, 2H),7.65 (m, 4H), 7.5 (t, 2H), 3.6 (br s), 1.57 (s, 6H).

3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine: ethylacetate/hexane gradient silica gel chromatography, trituration from 2:3ether:hexane, Rf=0.14 (1:1 ethyl acetate:hexane). ¹H-NMR (300 MHz,CDCl₃) δ (ppm): 7.48 (s, 1H), 7.3 (s, 1H), 6.58 (s, 1H), 3.96 (s, 3H),3.92 (s, 3H), 3.65 (br s, 2H), 1.52 (s, 6H).

3-(2,4-Dichloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine. ¹H-NMR(300 MHz, CDCl₃) δ (ppm): 7.5 (s, 1H), 7.45 (d, 1H), 7.35 (d, 1H), 3.53(s, 2H), 1.57 (s, 6H).

Method A-2

A mixture of indan-2-ylidene-hydrazine (250 mg) and3,5-dimethyl-benzaldehyde chlorooxime (314 mg) were mixed with CHCl₃ (10mL) and aqueous K₂CO₃ (6 mL, 0.167 g/mL) at 45° C. for a period of 4hours. The phases of the reaction mixture were diluted and partitioned,and the organic layer was dried and the solvent was evaporated in vacuo.Column chromatography of the crude product on silica gel using 10% ethylacetate in hexane yielded 0.52 g of3-(3,5-dimethyl-phenyl)-7,8-benzo-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-ylamine.An analytical sample was crystallized from CHCl₃/pentane. ¹H NMR (CDCl₃,300 MHz) δ (ppm): 7.37 (s, 2H), 7.22 (m, 4H), 7.11 (s, 1H), 3.6 (d, 2H),3.57 (br s, 2H), 3.32 (d, 2H), 2.36 (s, 6H).

Method B

A round bottom flask was charged with a solution of 20 g of K₂CO₃ in 50mL of water and cooled in an ice bath. Cyclopentylidene-hydrazine (7.9g, 80 mmol) in 25 mL of CH₂Cl₂ was added, followed by a drop-wiseaddition of benzohydroximoyl chloride (5 g, 0.032 mmol) in 25 mL ofCH₂Cl₂ over a period of 15 minutes. The mixture was stirred for severaldays, and allowed to warm to room temperature. Water (50 mL) and CH₂Cl₂(50 mL) were added and the phases were separated. The organic layer waswashed twice with 50 mL of water, dried over MgSO₄, and filtered. Thesolvent was removed in vacuo, leaving 9 g of a waxy solid.Crystallization from ethyl acetate/hexane provided 3.5 g (50%) of3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-ylamine after drying.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.72 ((m, 2H), 7.46 (m, 3H), 3.54 (s,2H), 2.1 (m, 2H), 2.0 (m, 2H), 1.85 (m, 2H), 1.8 (m, 2H).

The following additional oxadiazolines were prepared by method B:

3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-ylamine:crystals from hexanes, 9% yield. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.31((s, 2H), 7.1 (s, 1H), 3.5 (s, 2H), 2.35 (s, 6H), 1.85 (m, 2H), 1.48 (s,3H), 1.03 (t, 3H).

3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-ylamine:crystals from ethyl acetate, 20% yield. ¹H-NMR (300 MHz, CDCl₃) δ(ppm)-7.32 ((s, 2H), 7.1 (s, 1H), 3.55 (s, 2H), 2.35 (s, 6H), 2.1 (m,2H), 2.0 (m, 2H), 1.85 (m, 2H), 1.8 (m, 2H).

3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-ylamine:crystals. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.34 (s, 2H), 7.15 (s, 1H),4.0 (m, 2H), 3.87 (dt, 2H), 3.55 (br s, 2H), 2.36 (s, 6H), 2.1 (m, 2H),1.9 (br d, 2H).

3-Phenyl-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-ylamine: crystals fromCHCl₃/hexane after silica gel chromatography (0-15% ethyl acetate inhexanes), m.p. 168-9 C, Rf=0.5 (1:1 ethyl acetate:hexanes). ¹H-NMR (300MHz, CDCl₃) δ (ppm): 7.86 (m, 2H), 7.157 (m, 3H), 3.92 (m, 2H), 3.87(dt, 2H), 3.61 (br s, 2H), 2.1 (m, 2H), 1.85 (br d, 2H).

Method C

A solution of benzohydroximoyl chloride in CCl₄, was added drop-wiseover 15 minutes to a solution of isopropylidene-hydrazine (3.6 g, 50mmoles) and triethylamine (3.0 g, 50 mmoles) in 10 mL of CHCl₃. Stirringwas continued for 2 hours while the mixture was allowed to warm to roomtemperature. TLC indicated that the reaction was complete. The reactionmixture was washed three times with water, dried over MgSO₄, andfiltered. The solvent was removed in vacuo, and the product,5,5-Dimethyl-3-phenyl-[1,2,4]oxadiazol-4-ylamine, was recrystallizedfrom CHCl₃/hexane, resulting in 1.2 g at a yield of 20.9%. ¹H-NMR (300MHz, CDCl₃) δ (ppm): 7.79 (m, 2H), 7.5 (m, 3H), 3.55 (s, 2H), 1.55 (s,6H). Use of high quality hydroximoyl chloride and addition at 0° C.resulted in improved yields (40%).

The following additional oxadiazoline was prepared by method C:

3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine:purification by ethyl acetate/hexane gradient silica gel chromatography(Rf=0.5 2:1 hexane:ethyl acetate), or crystallization fromether/heptane. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.32 (s, 2H), 7.12 (s,1H), 3.52 (s, 2H), 2.35 (s, 6H), 1.53 (s, 6H).

TABLE 1 Optimization of [3 + 2] cycloaddition of benzonitrile N-oxideand isopropylidene-hydrazine.

Method Solvent Base Temp Time Yield Comments A CHCl₃/ K₂CO₃ 40-50° C. 2hr 40% faster, H₂O convenient B CHCl₃/ K₂CO₃ 0-40° C. 6 hr 45% productH₂O easier to purify C CH₂Cl₂ Et₃N 0-25° C. 3 hr 20- 24% D CHCl₃ Et₃N0-25° C. over- 40% impurities night appear after 3 hours E iso- Et₃N 25°C. 2 hr 18% pro- panol F CHCl₃ pyridine 25° C. 3 hr  7% G toluene/ K₂CO₃45° C. 4 hr 27% H₂O Notes: 1. Chlorooxime/CHCl₃ solution added tomixture of hydrazone and base, unless otherwise indicated. 2. Reactionsrun with acetone hydrazone of ca. 80% purity (azine comprises theremainder). 3. Product purified by trituration with 2% ether/hexanes,but can also be chromatographed on silica gel using 20% EtOAc inhexanes, Rf = 0.4 in 1:1 ethylacetate:hexanes.

1.13 Preparation of N-aroyl-4-amino-Δ²-1,2,4-oxadiazolines Method A:

4-Ethylbenzoyl chloride (78.4 mg, 0.466 mmol) and3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine (100 mg,0.444 mmol) were dissolved in 4 mL of ethyl acetate in a 20 mL vial.With magnetic stirring, an aqueous solution of K₂CO₃ (2 mL, 0.166 g/mL)was added, and the mixture was stirred at room temperature for 18-64hours. The reaction mixture was transferred to a separatory funnel. Theorganic phase was removed and evaporated to dryness under vacuum at roomtemperature and then at 50° C. for 30 minutes. The residue wastriturated with a solution of 10% ether in hexane (7 mL) for 3-8 hourswith magnetic stirring, and the resultant flocculent precipitate wasremoved and triturated again with 5% ether in hexane. Vacuum oven dryingat 50° C. for 30 minutes yieldedN-[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-ethyl-benzamidein 90% purity. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.72 (d, 2H), 7.6 (d,2H), 7.35 (d, 2H), 7.25 (d, 2H), 2.7 (q, 2H), 1.63 (s, 6H), 1.22 (t,3H). Some analogs were purified by trituration with pentane, hexane, orheptane, or alternatively, by column chromatography using an ethylacetate/hexane gradient.

Most N-aroyl-4-amino-Δ²-1,2,4-oxadiazolines were made by this method.

Method B:

To a solution of 5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-ylamine (0.5g, 2.6 mmol) in 5 mL of CH₂Cl₂, was added aqueous K₂CO₃ (0.54 g, 3.9mmol in 5 mL of water). The mixture was cooled in an ice bath, and asolution of 2-methyl-3-methoxybenzoyl chloride in 5 mL of CH₂Cl₂ wasadded to the reaction mixture. Stirring was continued at roomtemperature for several days. Water and CH₂Cl₂ were added, the phaseswere separated, and the organic layer was washed twice with water, oncewith brine, and dried over MgSO₄. The solution was filtered and thesolvent was removed in vacuo. Column chromatography on silica gelprovided high purityN-(5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-methoxy-2-methyl-benzamide,but in low yield. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.75 (s, 1H[NH]),7.70 (d, 2H), 7.4 (m, 3H), 7.1 (t, 1H), 6.85 (d, 1H), 6.6 (d, 1H), 3.78(s, 3H), 1:99 (s, 3H), 1.61 (s, 6H), m.p. 141-142° C. A major by-productwas 2-methyl-3-methoxybenzoyl anhydride.

Method C-1:

A 20% aqueous solution of NaOH (275 mg, 1.37 mmol) was added to asolution of3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine (200 mg,0.91 mmol) in 2 mL of toluene. 2-Ethyl-3-methoxybenzoyl chloride (199mg, 1 mmol) was then added to the mixture. The reaction was stirred atroom temperature for 2 hours, and then heated at 50° C. for 1 hour. TLCindicated 3 spots. Water, dilute NaOH, and CHCl₃ were then added to thereaction mixture. The organic phase was separated, dried over MgSO₄, andthe solvent removed in vacuo. The residue was triturated with 10% etherin hexane. Filtration and air-drying of the resultant solid provided 100mg (29% yield) ofN-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide,¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.42 (s, 2H), 7.11 (s, 1H), 7.11 (t,1H), 6.9 (d, 1H), 6.67 (d, 1H), 3.81 (s, 3H), 2.55 (q, 2H), 2.34 (s,6H), 1.7 (br s, 6H), 1.05 (t, 3H), Rf=0.5 (1:1 ethyl acetate:hexane).

Method C-2:

Approximately 18.5 mg (0.42 mmol) of5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl chloride were added to103 mg (0.5 mmol) of 5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-ylamine in2 mL of toluene in a 20 mL vial. Then 400 mg of a 20% aqueous NaOHsolution were added. The mixture was stirred at room temperature for 20hours, and then gently heated at 50° C. for 1 hour. The reaction mixturewas transferred to a separatory funnel with CHCl₃ and extracted withdilute NaHCO₃. The CHCl₃ extract was dried and evaporated to dryness.The residue was triturated with pentane to remove the toluene, and thentriturated with 5% ether-hexane. ¹H NMR indicated the intended product,5-ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid(5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide, at a purity levelof ca, 60%. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.42 (s, 2H), 7.11 (s, 1H),7.11 (t, 1H), 6.9 (d, 1H), 6.67 (d, 1H), 3.81 (s, 3H), 2.55 (q, 2H),2.34 (s, 6H), 1.7 (br s, 6H), 1.05 (t, 3H), Rf=0.5 (1:1 ethylacetate:hexane).

Method D:

A 5% aqueous solution of NaOH (4 mL) was added to a solution of3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine (90 mg)in 6 mL of toluene. 2-Ethyl-3-methoxybenzoyl chloride (140 mg) was thenadded to the mixture. The reaction was stirred at room temperature for30 hours. Water and CHCl₃ were added. The organic phase was separated,dried over MgSO₄, and the solvent was removed in vacuo. The residue wastriturated with 100 mL of 10% ether in hexane by magnetic stirring ofthe mixture in a vessel for 1 hour. Filtration and air-drying of theresultant solid provided 62 mg (40% yield) ofN-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide.¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7.42 (s, 2H), 7.11 (s, 1H), 7.11 (t,1H), 6.9 (d, 1H), 6.67 (d, 1H), 3.81 (s, 3H), 2.55 (q, 2H), 2.34 (s,6H), 1.7 (br s, 6H), 1.05 (t, 3H), Rf=0.5 (1:1 ethyl acetate:hexane).The filtrate contained an additional quantity of the intended product.

Method E:

To an ice-cold stirred solution of the5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-ylamine (0.5 g, 2.6 mmol) in 6mL of ethanol, was added an excess of 4-ethylbenzoyl chloride (4 mL),followed by aqueous NaOH (8%, 7 mL). The solution was stirred overnightand allowed to warm to room temperature. The mixture was diluted withwater and extracted with CH₂Cl₂. The combined CH₂Cl₂ extracts were driedover MgSO₄, and the solvent was removed in vacuo. The product,N-(5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-ethyl-benzamide, wasisolated by silica gel chromatography. ¹H-NMR (300 MHz, CDCl₃) δ (ppm):7.8 (m, 3H), 7.6 (d, 2H), 7.4 (m, 2H), 7.2 (d, 2H), 2.7 (q, 2H), 1.65(s, 6H), 1.2 (t, 3H).

TABLE 2 Optimization of amide formation between 2-ethyl-3-methoxybenzoylchloride and 3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine.

Time Temp Base Yield 4-5 hr 25° C. 20% NaOH 24% 2 days 25° C. 25% K₂CO₃20% 2 days 25° C. 50% K₂CO₃  8% 20-30 hr 25° C.  5% NaOH 40% 20+ hr 25°C.  3% NaOH >60%  1. Purification by trituration with 5-10%ether-hexanes gave 90-98% pure product. Higher ether content reducesyield but enhances purity. 2. In separate experiments with 3-NO₂oxadiazoline, K₂CO₃/CH₂Cl₂ gave at least 50% yields. 3. K₂CO₃/EtOH alsofound to be acceptable. (1 eq. oxadiazoline, 2 eq. ROCl, 2.5 eq. K₂CO₃).4. NaH/THF at 25° C. or NaH/THF/DMF at 25-75° C. for the unsubstitutedoxadiazoline and 4-ethylbenzoyl chloride results in only a trace ofproduct at best. Heating the two reactants neat at 150° C. for 2 hoursresults in a tar. 5. Powdered KOH in THF for the unsubstitutedoxadiazoline and 2-methyl-3-methoxybenzoyl chloride results in only atrace of product.

1.14 Preparation of aryl-[1.2.4]oxadiazol-4-yl-ureas from the Reactionof aryl-[1,2,4]oxadiazol-4-ylamines with Isocyantes

3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-ylamine (219 mg,1 mmol) and phenylisocyanate (131 mg, 1.1 mmol) were dissolved in 1 mLof toluene and stirred at room temperature for 2 hours. TLC indicated apartial reaction; therefore the mixture was heated at 50° C. for 2hours. The solvent was removed in vacuo and the residue was stirred in25 mL of 10% ether in hexane.1-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-ureawas recovered as a white precipitate (106 mg, 31% yield). ¹H-NMR (300MHz, CDCl₃) δ (ppm): 7.78 (s, 1H [NH]), 7.5 (d, 2H), 7.4 (t, 2H), 7.3(s, 2H), 7.2 (t, 1H), 7.15 (s, 1H), 6.4 (s, 1H [NH]), 2.28 (s, 6H), 1.73(s, 3H), 1.54 (s, 3H).

Preparation ofN-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-methoxy-2-methyl-benzamide(RG-120045)

To a 500 mL, 3-neck flask equipped with a magnetic stirrer, and chilledin an ice water bath, were added 25 mL of CH₂Cl₂ (significantly greaterquantities can be used) and 22.5 g (450 mmol) of hydrazine hydrate,followed by a solution of 31.5 g of K₂CO₃ dissolved in 60 mL of water.Over a period of 30 minutes, a solution of 31 g (168 mmol) of 2-methyl,3-methoxybenzoyl chloride dissolved in 50 mL of CH₂Cl₂ was added, whilekeeping the temperature below 5° C. The reaction mixture was allowed towarm to room temperature and then stirred for an additional 2 hours.Water (100 mL) and chloroform (150 mL) were added, the mixture wasshaken in a separatory funnel, and an inorganic precipitate was filteredoff. The organic layer was dried over MgSO₄ and the solvent removed invacuo to leave 30 g of crude product hydrazide. This material wasslurried with heptane for 4 hours (pentane slurry gives comparableresults). Filtration and residual solvent evaporation yielded 13 g of3-methoxy-2-methyl-benzoic acid hydrazide, containing ca. 10% ofdiacylated material. The product could be further purified byprecipitation with hot CHCl₃/hexane. ¹H NMR (300 MHz, CDCl₃) δ (ppm):7.2 (t, 1H), 6.95 (br s, 1H), 6.9 (m, 2H), 4.15 (br s, 2H), 3.84 (s,3H), 2.27 (s, 3H).

3-methoxy-2-methyl-benzoic acid hydrazide (1.1 g) was dissolved in 10 mLof acetone in a 25 mL round bottom flask. 2 drops of acetic acid wereadded and the reaction was stirred at room temperature for 10 minutes.The vessel was placed in a refrigerator for 1 hour and the product,3-methoxy-2-methyl-benzoic acid isopropylidene-hydrazide, was filteredoff and dried. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.2 (t, 1H), 7.02 (d,1H), 6.94 (d, 1H), 3.85 (s, 3H), 2.33 (s, 3H), 2.18 (s, 3H, benzylic),1.88 (s, 3H).

3-Methoxy-2-methyl-benzoic acid isopropylidene-hydrazide (1.71 g, 7.77mmol) and 3,5-dimethyl-benzaldehyde chlorooxime (2.29 g, 12.4 mmol),both as CHCl₃ solutions (total volume 40 mL), were added to a 250 mLround bottom flask equipped with a magnetic stirrer. An aqueous K₂CO₃solution (5 g in 30 mL) was added, the vessel was placed in a 50° C.water bath, and the reaction was stirred vigorously for 24 hours. Thereaction was monitored by ¹H NMR. Chloroform (100 μl) and water (50 mL)were added, and the mixture was shaken in a separatory funnel. Theorganic phase was separated, dried, and the solvent was removed invacuo. Column chromatography on silica gel using a gradient of 10-30%ethyl acetate in hexane yielded 1.07 g ofN-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-methoxy-2-methyl-benzamide(32%). An analytical sample was obtained by crystallization fromCH₂Cl₂/hexane under refrigeration. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.4(s, 2H), 7.1 (t, 1H), 7.07 (s, 1H), 6.85 (d, 1H), 6.65 (d, 1H), 3.78 (s,3H), 2.30 (s, 6H), 2.07 (s, 3H), 1.65 (s, 6H).

TABLE 3 Summary of the reaction conditions explored. YieldHydrazone:Oxime (by ¹H Base/Solvent Time/Temp Chloride NMR) 1.25 eq.Et₃N, CHCl₃ Overnight, 25° C. 1:1  5-10% 1.25 eq. Et₃N, CHCl₃  4 hr, 45°C. 1:2 20-30% 17% aq.  3 hr, 45° C. 1:1 20-25% K₂CO₃/CHCl₃ 17% aq.  5hr, 45° C. 1:1 25-30% K₂CO₃/CHCl₃ 1.8 eq. Et₃N,  2 hr, reflux   1:1.315-20% ClCH₂CH₂Cl K₂CO₃ (powder)/  2 hr, 60° C.   1:1.7 25-30%ClCH₂CH₂Cl K₂CO₃ (powder) +  4 hr, reflux   1:1.5 10% MgSO₄ (powder) 17%aq. K₂CO₃/ 24 hr, 60° C. 1:3 32% CHCl₃ (isolated) In general, K₂CO₃ asbase gave cleaner products compared with the Et₃N.

1.16 Preparation ofN-[3-(3,5-dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide(RG-120086)

3-methoxy-2-methyl-benzoic acid hydrazide (4.14 g, 23 mmol) wassuspended in a mixture of 80 mL of ether, 40 mL of CH₂Cl₂ and 2 drops ofacetic acid. A solution of tetrahydro-pyran-4-one (2.3 g, 23 mmol) in 20mL of CH₂Cl₂ and 2-3 mL of methanol were added, and the mixture wasrefluxed for 10 minutes. The reaction mixture was allowed to cool andwas concentrated to about 80 mL. Pentane (100 mL) was added, resultingin the formation of a precipitate. The suspension was chilled in afreezer and filtered to yield 5.04 g of 3-methoxy-2-methyl-benzoic acid(tetrahydro-pyran-4-ylidene)-hydrazide as a tan, flocculent,semi-crystalline material. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.2 (t, 1H),6.98 (d, 1H), 6.95 (d, 1H), 3.9 (t, 2H), 3.86 (s, 3H), 3.8 (t, 2H), 2.65(t, 2H), 2.42 (t, 2H), 2.35 (s, 3); Rf=0.15 (3:1 ethyl acetate:hexane).

3,5-dimethyl-benzaldehyde chlorooxime (1.84 g, 4 mmol) was dissolved in30 μL of CHCl₃ in a round bottom flask. An aqueous solution of K₂CO₃ (25mL, 0.166 g/mL) was added, followed by 1.05 g of3-methoxy-2-methyl-benzoic acid (tetrahydro-pyran-4-ylidene)-hydrazide.The reaction was stirred overnight at 55-60° C. Water and CH₂Cl₂ (50 mL)were added, and the mixture was shaken in a separatory funnel. Theaqueous phase was removed and extracted once with CHCl₃ (25 mL). Theorganic phases were combined, washed once with dilute K₂CO₃, and driedover MgSO₄. The solvent was removed in vacuo to yield 2.5 g of crudeproduct. This material was triturated twice with 10% ether in hexane andthen once with 25% ether in hexane. The solids were collected andfiltered, yielding 720 mg ofN-[3-(3,5-dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4.5]dec-2-en-4-yl]-3-methoxy-2-methyl-benzamideat 80% purity and a 49% yield. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 8.5 (s,1H [NH]), 7.4 (s, 2H), 7.1 (m, 1H), 7.1 (s, 1H), 6.9 (d, 1H), 6.65 (d,1H), 3.9 (m, 4H), 3.8 (s, 3H), 2.4 (m, 4H), 2.35 (s, 6H), 2.05 (s, 3H).

1.17 Preparation ofN-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,5]-7,8-benzo-dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide(RG-120037)

3-Methoxy-2-methyl-benzoic acid hydrazide (1.0 g) and β-tetralone (0.9g) were mixed in 4 ml of methanol with 1 drop of acetic acid at roomtemperature for 10 minutes. Approximately 10 mL of ether were added andthe mixture was refrigerated. Crystals of 3-methoxy-2-methyl-benzoicacid (3,4-dihydro-1H-naphthalen-2-ylidene)-hydrazide formed, which werecollected by filtration (0.85 g). ¹H NMR (300 MHz, CDCl₃) δ ppm):6.8-7.3 (m, 4H), 3.75+3.8 (2 s, 3H), 3.45+3.7 (2 s, 2H), 2.85 (t, 2H),2.4 (t, 2H), 2.27+2.25 (2 s, 3H); multiple conformers; Rf=0.56 (3:1ethyl acctate:hexane); m.p.=138° C.; m.p. of 3-methoxy-2-methyl-benzoicacid hydrazide=113-116° C.

3-Methoxy-2-methyl-benzoic acid(3,4-dihydro-1H-naphthalen-2-ylidene)-hydrazide (1.25 g) was mixed with3,5-dimethyl-benzaldehyde chlorooxime (1.68 g) and 3.1 g oftriethylamine in 5 mL of DMF in a round bottom flask, causing thereaction mixture to turn red immediately. Water was added to thereaction mixture and extracted with ether to yield 2.05 g of crudeproduct, which was then chromatographed twice on alumina using ahexane/ethyl acetate/methanol gradient. The intended product,N-[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4.5]-7,8-benzo-dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide,eluted with solvent compositions ranging from 60:40 ethyl acctate:hexaneto 97:3 ethyl acetate:methanol. ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.1 (s,2H), 7.05 (s, 1H), 7.03 (m, 2H), 7.0 (m, 2H), 6.95 (m, 2H), 6.75 (m,1H), 4.8 (br, 1H [NH]), 3.71 (s, 3H), 2.8 (m, 1H), 2.65 (m, 1H), 2.22(m, 4H), 2.2 (s, 6H), 2.1+2.05 (2 s, 3H). An analogous reaction inCHCl₃/aqueous K₂CO₃ at 60° C. overnight or in acetonitrile with Koenig'sbase at reflux gave little or none of the desired product.

TABLE 4 Physical Characterization of Compounds Compound NMR frequencySolvent R¹ R⁴ R¹ + R⁴ R² + R³ NH RG-120111 300 MHz CDCl3 7.95 (d, 2H),7.35 (s, 2H), 1.62 (s, 6H) 9.15 (s, 1H) 7.65 (m, 1H), 7.03 (s, 1H), 7.55(m, 2H) 2.26 (s, 6H) RG-120056 300 MHz CDCl3 6.95 (m, 1H), 7.35 (s, 2H),1.56 (s, 6H) 7.7 (s, 1H), 7.3 (br, 3H), 7.1 (s, 1H), 2.29 (s, 6.0 (s,1H) 2.38 (s, 3H) 6H) RG-120072 300 MHz CDCl3 7.5 (d, 2H), 7.3 (s, 2H),1.73 (s, 3H), 7.78 (s, 1H), 7.4 (t, 2H), 7.2 (t, 7.15 (s, 1H), 2.28 (s,1.54 (s, 3H) 6.4 (s, 1H) 1H) 6H) RG-120075 300 MHz CDCl3 8.1 (d, 1H),7.4 (s, 2H), 1.56 (s, 6H) 9.0 (s, 1H) 7.5 (m, 1H), 7.1 (m, 7.05 (s, 1H),2.27 (s, 1H), 6.95 (d, 6H) 1H), 3.83 (s, 3H) RG-120091 300 MHz CDCl3 8.4(d, 1H), 7.4 (s, 2H), 1.57 (s, 6H) 8.45 (s, 1H) 8.0 (d, 1H), 7.75 (s,7.1 (s, 1H), 2.29 (s, 1H), 7.65 (t, 1H) 6H) RG-120098 300 MHz CDCl3 8.1(m, 1H), 7.45 (s, 2H), 1.56 (s, 6H) 7.6 (m, 2H), 7.1 (m, 7.15 (s, 1H),1H) 2.37 (s, 6H) RG-120077 300 MHz CDCl3 7.50 (s, 1H), 7.41 (s, 2H),1.57 (s, 6H) 7.45 (s, 1H), 7.05 (s, 1H), 7.35 (m, 1H), 2.28 (s, 6H) 7.05(s, 1H), 2.36 (s, 3H) RG-120060 300 MHz CDCl3 7.55 (d, 2H), 7.4 (s, 2H),1.56 (s, 6H) 7.25 (d, 2H), 7.05 (s, 1H), 2.3 (s, 2.7 (q, 2H), 1.25 (t,6H) 3H) RG-120024 300 MHz CDCl3 7.55 (d, 2H), 7.4 (s, 2H), 1.56 (s, 6H)7.4 (d, 2H) 7.05 (s, 1H), 2.28 (s, 6H) RG-120080 300 MHz CDCl3 7.11 (t,1H), 7.42 (s, 2H), 1.7 (br s, 6H) 6.9 (d, 1H), 6.67 (d, 7.11 (s, 1H),1H), 3.81 (s, 2.34 (s, 6H) 3H), 2.55 (q, 2H), 1.05 (t, 3H) RG-120002 300MHz CDCl3 7.55 (s, 1H), 7.4 (s, 2H), 1.57 (s, 6H) 7.2-7.4 (m, 3H) 7.05(s, 1H), 2.29 (s, 6H) RG-120015 300 MHz CDCl3 7.6 (m, 2H), 8.07 (s, 1H),1.66 (s, 6H) 7.5 (m, 1H), 7.4 (t, 7.97 (d, 1H), 2H) 7.7 (d, 1H), 7.55(m, 1H) RG-120148 300 MHz CDCl3 7.55 (d, 2H), 8.1 (s, 1H), 1.64 (s, 6H)7.75 (br, 1H) 7.2 (d, 2H), 2.68 (q, 7.97 (d, 1H), 7.7 (d, 2H), 1.21 (t,3H) 1H), 7.5 (d, 1H) RG-120022 300 MHz CDCl3 7.15 (t, 1H), 7.87 (d, 2H),1.65 (br s, 6H) 7.20 (s, 1H) 6.9 (d, 1H), 6.62 (d, 7.3 (d, 2H) 1H), 3.80(s, 3H), 2.50 (br s, 2H), 0.97 (t, 3H) RG-120094 300 MHz CDCl3 7.42 (s,1H), 7.85 (d, 2H), 1.62 (s, 6H) 7.27 (s, 1H), 7.22 (d, 2H) 6.55 (m, 1H)RG-120160 300 MHz CDCl3 7.85 (m, 1H), 7.67 (d, 2H), 1.48 (s, 6H) 6.85(s, 1H) 7.25 (m, 2H), 7.18 (d, 2H) 7.05 (m, 2H), 3.48 (s, 2H) RG-120066300 MHz CDCl3 7.32 (m, 3H), 7.77 (d, 2H), 1.66 (s, 6H) 7.95 (s, 1H) 7.18(m, 2H), 7.25 (d, 2H) 4.44 (s, 2H), 3.98 (s, 2H) RG-120088 300 MHz CDCl37.3-7.4 (m, 4H), 7.2 (s, 2H), 1.56 (s, 6H) 5.95 (s, 1H) 6.15 (m, 1H),7.1 (s, 1H), 2.27 (s, 4.6 (dd, 1H), 6H) 4.45 (dd, 1H) RG-120029 300 MHzCDCl3 7.25 (m, 4H), 7.75 (d, 2H), 1.55 (s, 6H) 7.9 (s, 1H) 7.05 (t, 1H),6.78 (d, 2H) 4.52 (s, 2H) RG-120109 300 MHz CDCl3 7.95 (m, 1H), 7.85 (d,2H), 1.66 (s, 6H) 7.8 (m, 1H), 7.21 (d, 2H) 7.55 (m, 1H), 7.47 (m, 2H)RG-120033 300 MHz CDCl3 7.62 (d, 2H), 7.82 (d, 2H), 1.65 (s, 6H) 7.55(t, 1H), 7.21 (d, 2H) 7.4 (t, 2H) RG-120055 300 MHz CDCl3 7.55 (d, 2H),7.8 (d, 2H), 1.61 (s, 6H) 7.65 (s, 1H) 7.25 (d, 2H), 7.21 (d, 2H) 2.7(q, 2H), 1.22 (t, 3H) RG-120147 300 MHz CDCl3 7.57 (m, 3H), 7.52 (m,1H), 1.66 (s, 6H) 8.05 (s, 1H) 7.42 (m, 2H) 7.5 (m, 1H), 7.05 (t, 1H),6.97 (d, 1H), 3.89 (s, 3H) RG-120062 300 MHz CDCl3 7.5 (d, 1H), 1.62 (s,6H) 8.55 (br s, 1H) 7.5 (t, 1H), 7.07 (t, 1H), 7.02 (d, 1H), 3.95 (s,3H) RG-120026 300 MHz CDCl3 7.5 (d, 2H), 7.6 (d, 1H), 1.65 (s, 6H) 8.05(s, 1H) 7.2 (d, 2H), 2.65 (q, 7.45 (t, 1H), 7.05 (t, 2H), 1.22 (t, 3H)1H), 7.0 (d, 1H), 3.91 (s, 3H) RG-120070 300 MHz CDCl3 7.45 (s, 1H),8.07 (s, 1H), 1.65 (s, 6H) 7.85 (s, 1H) 7.2 (d, 1H), 6.5 (m, 7.95 (d,1H), 1H) 7.68 (d, 1H), 7.55 (t, 1H) RG-120093 300 MHz CDCl3 7.2 (t, 1H),8.1 (s, 1H), 1.7 (br s, 6H) 6.9 (d, 1H), 6.65 (d, 8.05 (d, 1H), 7.75 (d,1H), 3.80 (s, 1H), 7.6 (t, 1H) 3H), 2.47 (br s, 2H), 0.97 (t, 3H)RG-120006 300 MHz CDCl3 7.35 (m, 3H), 8.0 (s, 1H), 1.57 (s, 6H) 7.18 (m,2H), 7.92 (d, 1H), 7.75 (d, 4.45 (s, 2H), 1H), 7.57 (t, 1H) 3.95 (s, 2H)RG-120108 300 MHz CDCl3 7.85 (m, 2H), 8.1 (s, 1H), 1.57 (s, 6H) 7.8 (s,1H), 7.97 (d, 1H), 7.7 (d, 7.45 (m, 2H) 1H), 7.55 (m, 1H) RG-120021 300MHz CDCl3 7.35 (m, 3H), 7.9 (s, 1H), 1.48 (br s, 6H) 8.1 (s, 1H) 7.05(m, 2H), 7.85 (d, 1H), 7.7 (d, 3.46 (s, 2H) 1H), 7.5 (t, 1H) RG-120163300 MHz CDCl3 7.25 (d, 1H), 8.05 (s, 1H), 1.57 (s, 6H) 7.9 (s, 1H) 7.01(t, 1H), 7.95 (d, 1H), 7.79 (d, 1H), 7.7 (d, 1H), 7.55 (t, 4.5 (s, 2H)1H) RG-120059 300 MHz CDCl3 8.1 (s, 1H), 1.57 (s, 6H) 3.55 (br s, 1H)7.95 (d, 1H), 7.75 (d, 1H), 7.57 (t, 1H) RG-120001 300 MHz CDCl3 7.75(m, 1H), 8.0 (s, 1H), 1.71 (s, 3H), 7.37 (m, 2H), 7.8 (m, 1H), 7.55 (d,1.53 (s, 3H) 7.17 (m, 2H) 1H), 7.47 (m, 1H) RG-120153 300 MHz CDCl3 4.05(q, 2H), 8.1 (s, 1H), 3.55 (br s, 1H) 2.55 (br, 2H), 7.95 (d, 1H), 7.75(d, 2.30 (m, 2H), 1H), 7.57 (t, 1H) 1.57 (s, 6H), 1.2 (t, 3H) RG-120018300 MHz CDCl3 7.35 (m, 3H), 7.5 (m, 1H), 1.59 (s, 6H) 8.4 (s, 1H) 7.2(m, 2H), 7.4 (m, 1H), 7.02 (t, 4.4 (s, 2H), 3.95 (s, 1H), 6.85 (d, 2H)1H), 3.63 (s, 3H) RG-120057 300 MHz CDCl3 7.25 (m, 2H), 7.55 (d, 1H),1.57 (s, 6H) 8.4 (s, 1H) 6.75 (m, 3H), 7.4 (t, 1H), 7.05 (m, 4.45 (s,2H) 2H), 3.77 (s, 3H) RG-120025 300 MHz CDCl3 7.45 (m, 2H), 7.5 (m, 2H),1.8 (s, 3H), 7.75 (s, 1H), 7.35 (m, 2H), 7.05 (m, 2H), 1.5 (s, 3H) 6.35(s, 1H) 6.97 (m, 1H) 3.83 (s, 3H) RG-120122 300 MHz CDCl3 7.5 (d, 2H),7.75 (d, 2H), 1.59 (s, 6H) 6.55 (s, 1H) 7.4 (m, 2H), 7.2 (t, 7.3 (d, 2H)1H) RG-120047 300 MHz CDCl3 7.9 (m, 2H), 7.5 (m, 1H), 1.68 (s, 6H) 7.6(m, 1H), 7.5 (m, 7.4 (m, 1H), 2H) 7.05 (m, 1H), 7.0 (m, 1H), 3.95 (s,3H) RG-120144 300 MHz CDCl3 4.1 (q, 1H), 7.5 (m, 1H), 1.57 (s, 6H)2.2-2.7 (m, 4H), 7.4 (m, 1H), 1.2 (t, 3H) 7.05 (m, 1H), 7.0 (m, 1H), 3.9(s, 3H) RG-120127 300 MHz CDCl3 7.25 (m, 2H), 7.3 (d, 1H), 1.53 (s, 6H)7.9 (s, 1H) 7.05 (t, 1H), 7.2 (s, 1H), 6.8 (d, 6.8 (d, 2H), 4.52 (s,1H), 6.0 (s, 2H) 2H) RG-120017 300 MHz CDCl3 7.5 (m, 2H), 7.22 (s, 1H),1.73 (s, 6H) 7.4 (m, 2H), 7.2 (d, 1H), 6.8 (d, 7.15 (m, 1H) 1H), 5.99(s, 2H) RG-120140 300 MHz CDCl3 7.85 (m, 3H), 7.3 (d, 1H), 1.64 (s, 6H)7.45 (m, 2H) 7.25 (s, 1H), 6.8 (d, 1H), 5.96 (s, 2H) RG-120083 300 MHzCDCl3 4.1 (q, 2H), 7.2 (m, 2H), 1.56 (s, 6H) 2.6 (br, 2H), 2.3 (t, 6.8(d, 1H), 6.02 (s, 2H), 1.25 (t, 3H) 2H) RG-120156 300 MHz CDCl3 7.7 (m,2H), 7.3 (m, 2H), 1.62 (s, 6H) 7.55 (t, 1H), 6.8 (d, 1H), 5.98 (s, 7.45(t, 2H) 2H) RG-120012 300 MHz CDCl3 7.4 (s, 1H), 7.6 (d, 1H), 1.65 (s,6H) 8.35 (s, 1H) 7.5 (s, 1H), 6.5 (m, 7.45 (m, 1H), 7.05 (t, 1H) 1H),6.95 (d, 1H), 3.95 (s, 3H) RG-120061 300 MHz CDCl3 7.6 (d, 2H), 7.3 (m,2H), 1.62 (s, 6H) 7.25 (d, 2H), 2.7 (q, 6.8 (d, 1H), 5.98 (s, 2H), 1.22(t, 3H) 2H) RG-120016 300 MHz CDCl3 7.05 (t, 1H), 7.65 (d, 1H), 1.7 (s,6H) 7.4 (s, 1H) 6.9 (d, 1H), 6.45 (d, 7.45 (t, 1H), 1H), 3.83 (s, 7.1(t, 1H), 6.97 (d, 3H), 2.4 (q, 2H), 1H), 3.80 (s, 3H) 0.95 (t, 3H)RG-120157 300 MHz CDCl3 7.2 (m, 3H), 7.5 (m, 1H), 1.5 (s, 6H) 6.9 (m,2H), 3.45 (s, 7.4 (m, 1H), 7.0 (m, 2H) 1H), 6.8 (d, 1H), 3.57 (s, 3H)RG-120149 300 MHz CDCl3 7.45 (s, 1H), 7.3 (d, 1H), 1.60 (s, 6H) 7.8 (s,1H) 7.22 (m, 1H), 7.25 (s, 1H), 6.8 (d, 6.5 (m, 1H) 1H), 5.98 (s, 2H)RG-120081 300 MHz CDCl3 7.3 (m, 3H), 7.1 (m, 1H), 1.45 (s, 6H) 7.15 (m,2H), 6.85 (s, 1H), 3.5 (s, 2H) 6.8 (d, 1H), 6.02 (s, 2H) RG-120145 300MHz CDCl3 7.35 (m, 3H), 7.25 (m, 2H), 1.55 (s, 6H) 7.18 (m, 2H), 6.81(d, 1H), 4.45 (s, 2H), 6.01 (s, 2H) 4.0 (s, 2H) RG-120076 300 MHz CDCl37.1 (t, 1H), 7.8 (m, 2H), 2.5 (br s, 2H), 6.9 (d, 1H), 6.6 (d, 7.45 (m,3H) 1.9 (br s, 2H), 1H), 3.80 (s, 3H) 1.65 (br s, 3H), 1.15 (br s, 3H)RG-120100 300 MHz CDCl3 7.45 (m, 1H), 7.8 (m, 2H), 1.9 (m, 2H), 7.2 (m,1H), 7.4 (m, 3H) 1.57 (s, 3H), 6.5 (m, 1H) 1.13 (t, 3H) RG-120146 300MHz CDCl3 3.46 (s, 2H) 7.8-7.0 (m, 10H) 1.8 (m, 2H), 6.85 (s, 1H) 1.38(br s, 3H), 1.0 (m, 3H) RG-120154 300 MHz CDCl3 7.3 (m, 3H), 7.75 (d,2H), 1.85 (m, 2H), 7.15 (m, 2H), 7.45 (m, 3H) 1.51 (s, 3H), 4.39 (s,2H), 1.09 (t, 3H) 3.96 (s, 2H) RG-120103 300 MHz CDCl3 7.3 (m, 2H), 7.7(d, 2H), 1.85 (q, 2H), 7.9 (s, 1H) 7.0 (t, 1H), 6.85 (d, 7.4 (m, 3H)1.47 (s, 3H), 2H), 4.49 (s, 2H) 1.08 (t, 3H) RG-120095 300 MHz CDCl37.1-7.9 (m, 10H) 1.97 (m, 2H), 6.3 (br s, 1H) 1.52 (s, 3H), 1.15 (t, 3H)RG-120133 300 MHz CDCl3 7.8 (m, 5H), 1.95 (m, 2H), 7.4 (m, 5H) 1.61 (s,3H), 1.15 (t, 3H) RG-120118 300 MHz CDCl3 7.6 (d, 2H), 7.8 (d, 2H), 1.95(m, 2H), 7.5 (m, 1H), 7.4 (m, 7.4 (m, 3H) 1.59 (s, 3H), 2H) 1.15 (t, 3H)RG-120137 300 MHz CDCl3 7.6 (d, 2H), 7.8 (m, 2H), 1.95 (m, 2H), 7.2 (d,2H), 2.7 (q, 7.45 (m, 3H) 1.15 (t, 3H), 2H), 1.22 (t, 3H) 1.58 (s, 3H)RG-120058 300 MHz CDCl3 7.1 (t, 1H), 7.67 (s, 1H), 1.65 (s, 6H) 6.9 (d,1H), 6.6 (d, 7.27 (s, 1H), 1H), 3.95 (s, 6.52 (s, 1H), 3H), 2.45 (q,3.86 (s, 3H), 2H), 1.0 (t, 3H) 3.81 (s, 3H) RG-120102 300 MHz CDCl3 7.45(s, 1H), 7.6 (s, 1H), 1.62 (s, 6H) 8.1 (s, 1H) 7.15 (m, 1H), 7.3 (s,1H), 6.5 (m, 6.55 (m, 1H) 1H), 3.94 (s, 3H), 3.91 (s, 3H) RG-120078 300MHz CDCl3 7.35 (m, 2H), 7.5 (s, 1H), 1.49 (s, 6H) 7.1 (m, 3H), 7.3 (s,1H), 6.3 (s, 3.44 (s, 2H) 1H), 3.96 (s, 3H), 3.62 (s, 3H) RG-120110 300MHz CDCl3 7.37 (m, 3H), 7.55 (s, 1H), 1.57 (s, 6H) 8.2 (s, 1H) 7.22 (m,2H), 7.35 (s, 1H), 4.45 (s, 2H), 6.45 (s, 1H), 3.97 (s, 2H) 3.93 (s,3H), 3.67 (s, 3H) RG-120079 300 MHz CDCl3 7.22 (m, 2H), 7.55 (s, 1H),1.56 (s, 6H) 7.05 (t, 1H), 7.3 (s, 1H), 6.25 (s, 6.75 (d, 2H), 1H), 3.89(s, 4.47 (s, 2H) 3H), 3.77 (s, 3H) RG-120114 300 MHz CDCl3 7.5 (m, 2H),7.7 (s, 1H), 1.76 (s, 3H), 6.2 (s, 1H) 7.35 (m, 2H), 7.45 (s, 1H), 6.5(s, 1.52 (s, 3H) 7.1 (t, 1H) 1H), 3.93 (s, 3H), 3.82 (s, 3H) RG-120129300 MHz CDCl3 7.9 (m, 3H), 7.8 (s, 1H), 1.6 (s, 6H) 7.45 (m, 2H) 7.65(s, 1H), 6.5 (s, 1H), 3.95 (s, 3H), 3.90 (s, 3H) RG-120038 300 MHz CDCl34.1 (m, 2H), 7.5 (s, 1H), 1.54 (s, 6H) 2.2-2.7 (m, 4H), 7.4 (s, 1H), 6.5(s, 1.2 (t, 3H) 1H), 3.95 (s, 3H), 3.90 (s, 3H) RG-120096 300 MHz CDCl37.6 (m, 2H), 7.8 (s, 1H), 1.64 (s, 6H) 7.5 (m, 1H), 7.4 (m, 7.6 (s, 1H),6.5 (s, 1H) 1H), 3.91 (s, 3H), 3.89 (s, 3H) RG-120135 300 MHz CDCl3 7.55(d, 2H), 7.8 (s, 1H), 1.63 (s, 6H) 7.25 (d, 2H), 7.65 (s, 1H), 6.5 (s,2.7 (q, 2H), 1.23 (t, 1H), 3.91 (s, 3H) 3H), 3.89 (s, 3H) RG-120023 300MHz CDCl3 7.15 (t, 1H), 7.37 (d, 1H), 1.65 (br s, 6H) 7.9 (d, 1H), 6.65(d, 7.2 (s, 1H), 7.87 (d, 1H), 3.81 (s, 1H), 6.01 (s, 2H) 3H), 2.6 (q,2H), 1.05 (t, 1H) RG-120037 300 MHz CDCl3 7.03 (m, 2H), 7.1 (s, 2H), 7.0(m, 2H), 9.35 (s, 1H), 6.75 (m, 1H), 7.05 (s, 1H), 2.2 (s, 6.95 (m, 2H),4.8 (br, 1H) 3.71 (s, 3H), 2.1, 6H) 2.8 (m, 1H), 2.05 (2s, 3H) 2.65 (m,1H), 2.22 (m, 4H) RG-120086 300 MHz CDCl3 7.1 (m, 1H), 7.4 (s, 2H), 3.9(m, 4H), 8.5 (s, 1H) 6.9 (d, 1H), 6.65 (d, 7.1 (s, 1H), 2.35 (s, 2.4 (m,4H) 1H), 3.8 (s, 3H), 6H) 2.05 (s, 3H) RG-120051 300 MHz CDCl3 7.5 (d,2H), 7.4 (d, 2H), 2.0 (m, 2H), 8.8 (br d, 1H), 7.35 (m, 1H), 7.2 (m, 7.1(s, 1H), 2.27 (s, 1.75, 1.6, 6.25 (br t, 1H) 2H) 6H) 1.5 (3s, 3H), 1.2,1.1 (2d, 3H) RG-120161 300 MHz CDCl3 7.85 (m, 2H), 7.4 (s, 2H), 1.95 (m,2H), 7.6 (s, 1H) 7.8 (s, 1H), 7.05 (s, 1H), 2.27 (s, 1.60 (s, 3H), 7.45(m, 2H) 6H) 1.15 (t, 3H) RG-120126 300 MHz CDCl3 4.05 (q, 2H), 7.3 (s,2H), 1.85 (m, 2H), 2.55 (br, 2H), 7.1 (s, 1H), 2.31 (s, 1.52 (s, 3H),2.37 (m, 2H), 6H) 1.1 (t, 3H) 1.22 (t, 3H) RG-120004 300 MHz CDCl3 7.3(s, 2H), 1.9 (q, 2H), 7.1 (s, 1H), 2.3 (s, 1.53 (s, 3H), 1.1 (t, 6H) 3H)RG-120039 300 MHz CDCl3 7.6 (d, 1H), 7.4 (s, 2H), 1.95 (m, 2H), 7.5 (t,1H), 7.45 (d, 7.05 (s, 1H), 2.28 (s, 1.58 (s, 3H), 1H) 6H) 1.14 (t, 3H)RG-120128 300 MHz CDCl3 7.55 (d, 2H), 7.4 (s, 2H), 1.9 (m, 2H), 7.22 (d,2H), 7.05 (s, 1H), 2.23 (s, 1.57 (s, 3H), 2.7 (q, 2H), 1.2 (t, 6H) 1.15(t, 3H) 3H) RG-120162 300 MHz CDCl3 7.27 (m, 2H), 7.65 (d, 2H), 1.55 (s,6H) 7.9 (s, 1H) 7.05 (t, 1H), 7.35 (d, 2H) 6.8 (d, 2H), 4.5 (s, 2H)RG-120067 300 MHz CDCl3 7.8 (s, 1H), 7.75 (d, 2H), 1.73, 1.58 (2s, 6.5(s, 1H) 7.5 (d, 1H), 7.3 (m, 7.4 (d, 2H) 6H) 1H), 7.2 (t, 1H) RG-120087300 MHz CDCl3 7.85 (t, 2H), 7.75 (d, 2H), 1.65 (s, 6H) 7.8 (s, 1H), 7.45(t, 7.4 (d, 2H) 2H) RG-120164 300 MHz CDCl3 4.05 (q, 2H), 7.72 (d, 2H),1.55 (s, 6H) 2.6 (br, 2H), 2.35 (t, 7.35 (d, 2H) 2H), 1.25 (t, 3H)RG-120151 300 MHz CDCl3 7.65 (d, 2H), 7.7 (d, 2H), 1.65 (s, 6H) 3.5 (br,1H) 7.55 (t, 1H), 7.4 (d, 2H) 7.42 (m, 2H) RG-120035 300 MHz CDCl3 7.6(d, 2H), 7.72 (d, 2H), 1.63 (s, 6H) 7.25 (d, 2H), 2.7 (q, 7.35 (d, 2H)2H), 1.22 (t, 3H) RG-120045 300 MHz CDCl3 7.1 (t, 1H), 7.4 (s, 2H), 1.65(s, 6H) 6.85 (d, 1H), 6.65 (d, 7.07 (s, 1H), 2.30 (s, 1H), 3.78 (s, 6H)3H), 2.07 (s, 3H) RG-120042 300 MHz CDCl3 7.1 (t, 1H), 7.70 (d, 2H),1.61 (s, 6H) 7.75 (s, 1H) 6.85 (d, 1H), 6.6 (d, 7.4 (m, 3H) 1H), 3.78(s, 3H), 1.99 (s, 3H) RG-120115 200 MHz CDCl3 7.6 (d, 2H), 7.8 (m, 3H),1.65 (s, 6H) 7.2 (d, 2H), 2.7 (q, 7.4 (m, 2H) 2H), 1.2 (t, 3H) RG-120003300 MHz CDCl3 7.4 (s, 1H), 7.4 (s, 2H), 2.1 (m, 4H), 7.8 (s, 1H) 7.22(m, 1H), 6.5 (m, 7.05 (s, 1H), 2.29 (s, 1.85 (m, 4H) 1H) 6H) RG-120073300 MHz CDCl3 7.7 (d, 2H), 1.592 (s, 6H) 3.5 (br, 1H) 7.4 (d, 2H)RG-120005 300 MHz CDCl3 7.55 (d, 2H), 7.35 (s, 2H), 3.95 (br, 2H), 7.8(s, 1H) 7.2 (d, 2H), 2.7 (q, 7.05 (s, 1H), 3.85 (m, 2H), 2H), 1.2 (t,3H) 2.26 (s, 6H) 2.1 (br, 4H) RG-120008 300 MHz CDCl3 7.15 (t, 1H), 7.4(s, 2H), 4.02 (br s, 2H), 2.4 (br, 2H), 0.98 (t, 7.12 (s, 1H), 6.9 (d,3.9 (m, 2H), 3H) 1H), 6.6 (d, 1H), 2.1 (br, 4H) 2.33 (s, 6H) RG-120009300 MHz CDCl3 7.30 (br s, 3H), 7.35 (s, 2H), 3.95 (br s, 2H), 7.95 (s,1H) 7.12 (br s, 2H), 7.15 (s, 1H), 3.85 (m, 2H), 4.39 (s, 2H), 2.32 (s,6H) 2.0 (br s, 4H) 3.97 (s, 2H) RG-120011 300 MHz CDCl3 7.62 (m, 2H),7.45 (s, 2H), 2.12 (br s, 4H), 7.5 (m, 3H) 7.05 (s, 1H), 1.85 (br s, 4H)2.28 (s, 6H) RG-120013 300 MHz CDCl3 7.27 (m, 2H), 7.75 (d, 2H), 3.95(m, 2H), 7.9 (s, 1H) 7.0 (t, 1H), 7.45 (m, 3H) 3.85 (m, 2H), 6.75 (d,2H), 4.5 (s, 2.0 (br s, 4H) 2H) RG-120014 300 MHz CDCl3 4.05 (q, 2H),7.72 (m, 2H), 2.05 (br, 4H), 3.55 (s, 1H) 2.55 (br, 2H), 7.45 (m, 3H)1.8 (br, 4H) 2.35 (t, 2H), 1.23 (t, 3H) RG-120019 300 MHz CDCl3 7.6 (d,2H), 7.39 (s, 2H), 2.1 (br, 4H) 7.75 (s, 1H) 7.5 (m, 1H), 7.4 (m, 7.05(s, 1H), 2H) 2.27 (s, 6H) RG-120020 300 MHz CDCl3 7.1 (t, 1H), 7.9 (d,2H), 1.69 (br s, 6H) 6.9 (d, 1H), 6.65 (d, 7.82 (d, 1H), 7.7 (d, 1H),3.79 (s, 2H), 7.65 (d, 3H), 2.55 (q, 2H), 7.5 (m, 2H) 2H), 1.0 (t, 3H)RG-120027 300 MHz CDCl3 7.4 (m, 4H), 7.7 (d, 2H), 4.05 (m, 2H), 6.3 (s,1H) 7.2 (m, 1H) 7.5 (m, 3H) 3.85 (m, 2H), 2.05 (m, 4H) RG-120030 300 MHzCDCl3 7.67 (d, 2H), 7.5 (m, 2H), 1.95 (br s, 4H), 6.87 (s, 1H) 7.27 (m,1H), 7.35 (m, 3H) 1.8 (m, 4H) 7.05 (m, 2H), 3.47 (s, 2H) RG-120031 300MHz CDCl3 4.0 (q, 2H), 7.3 (s, 2H), 2.02 (br, 4H), 7.5 (s, 1H) 1.22 (t,3H), 2.6 (br, 7.05 (s, 1H), 2.31 (s, 1.8 (m, 4H) 2H), 2.35 (t, 2H) 6H)RG-120034 300 MHz CDCl3 7.35 (m, 3H), 7.75 (d, 2H), 3.97 (br, 2H), 8.0(s, 1H) 7.15 (m, 2H), 7.5 (m, 3H) 3.85 (m, 2H), 4.40 (s, 2H), 2.0 (br s,4H) 3.95 (s, 2H) RG-120040 300 MHz CDCl3 7.6 (d, 2H), 7.45 (s, 2H), 2.1(br, 4H), 7.22 (d, 2H), 2.7 (q, 7.05 (s, 1H), 1.8 (br, 4H) 2H), 1.22 (t,3H) 2.27 (s, 6H) RG-120041 300 MHz CDCl3 7.27 (t, 2H), 7.35 (s, 2H),2.05 (br s, 4H), 7.95 (s, 1H) 7.01 (t, 1H), 7.1 (s, 1H), 2.30 (s, 1.8(m, 4H) 6.8 (d, 2H), 4.5 (s, 6H) 2H) RG-120044 300 MHz CDCl3 7.4 (m, 5H)7.8 (d, 2H), 4.0 (br, 2H), 7.7 (s, 1H) 7.6 (d, 2H), 7.5 (m, 3.9 (t, 2H),2.1 (br, 1H) 4H) RG-120046 300 MHz CDCl3 7.3 (m, 3H), 7.25 (s, 2H), 1.95(br s, 4H), 7.05 (m, 2H), 7.1 (s, 1H), 2.28 (s, 1.75 (m, 4H) 3.5 (s, 2H)6H) RG-120048 300 MHz CDCl3 7.27 (m, 2H), 7.35 (s, 2H), 3.95 (m, 2H),7.9 (s, 1H) 7.0 (m, 1H), 7.1 (s, 1H), 2.31 (s, 3.85 (m, 2H), 6.75 (d,2H), 6H) 1.95 (br, 4H) 4.5 (s, 2H) RG-120049 300 MHz CDCl3 7.15 (m, 1H),7.45 (s, 2H), 2.15 (br s, 4H), 6.9 (d, 1H), 7.1 (s, 1H), 2.33 (s, 1.85(br s, 4H) 6.65 (d, 1H), 3.80 (s, 6H) 3H), 2.5 (q, 2H), 1.0 (t, 3H)RG-120050 300 MHz CDCl3 7.30 (m, 3H), 7.32 (s, 2H), 1.85 (m, 2H), 7.9(s, 1H) 7.12 (m, 2H), 7.1 (s, 1H), 2.32 (s, 1.5 (s, 3H), 4.4 (s, 2H),6H) 1.1 (t, 3H) 3.9 (s, 2H) RG-120052 300 MHz CDCl3 7.3 (m, 3H), 7.37(s, 2H), 2.0 (br s, 4H), 7.93 (s, 1H) 7.15 (m, 2H), 7.10 (s, 1H), 1.8(br s, 4H) 4.4 (s, 2H), 2.32 (s, 6H) 3.97 (s, 2H) RG-120054 300 MHzCDCl3 7.5 (m, 2H), 7.3 (s, 2H), 4.05 (m, 2H), 7.4 (m, 3H) 7.1 (s, 1H),2.27 (s, 3.9 (m, 2H), 6H) 2.2 (m, 2H), 2.1 (m, 2H) RG-120063 300 MHzCDCl3 7.72 (m, 2H), 2.05 (br, 4H), 3.55 (s, 1H) 7.4 (m, 3H) 1.8 (br, 4H)RG-120069 300 MHz CDCl3 7.1 (t, 1H), 7.41 (s, 2H), 1.95 (br s, 2H), 6.9(d, 1H), 6.65 (d, 7.1 (s, 1H), 2.33 (s, 1.65 (br s, 3H), 1H), 3.80 (s,6H) 1.15 (br t, 3H), 3H), 2.65 (br, 1.05 (br t, 3H) 2H), 1.05 (br t, 3H)RG-120071 300 MHz CDCl3 7.47 (m, 1H), 7.82 (m, 2H), 2.1 (br, 4H), 7.2(m, 1H), 7.4 (m, 3H) 1.8 (br, 4H) 6.52 (m, 1H) RG-120082 300 MHz CDCl37.45 (s, 1H), 7.75 (d, 2H), 1.62 (s, 6H) 7.2 (m, 1H), 6.5 (m, 7.4 (d,2H) 1H) RG-120084 300 MHz CDCl3 7.25 (m, 3H), 7.1 (s, 2H), 3.9 (m, 2H),7.0 (m, 2H), 6.87 (s, 1H), 2.30 (s, 3.8 (m, 2H), 3.45 (s, 2H) 6H) 1.90(br, 4H) RG-120089 300 MHz CDCl3 7.8 (d, 2H), 7.65 (d, 2H), 2.15 (br,4H), 7.4 (m, 3H) 7.5 (m, 1H), 7.4 (m, 1.85 (br, 4H) 2H) RG-120090 300MHz CDCl3 7.35 (s, 2H), 2.05 (m, 4H), 7.10 (s, 1H), 1.87 (m, 4H) 2.30(s, 6H) RG-120092 300 MHz CDCl3 7.10 (t, 1H), 7.82 (d, 1H), 2.1 (m, 2H),7.18 (s, 1H) 6.85 (d, 1H), 7.78 (d, 1H), 1.7-2.0 (m, 6H) 6.6 (d, 1H),3.80 (s, 7.45 (m, 3H) 3H), 2.5 (q, 2H), 1.0 (t, 3H) RG-120099 300 MHzCDCl3 7.73 (d, 2H), 6.9 (d, 1H), 1.7 (br, 6H) 7.2 (s, 1H) 7.42 (d, 2H),6.65 (d, 1H) 7.15 (t, 1H), 3.81 (s, 3H), 2.5 (br, 2H), 1.0 (t, 3H)RG-120106 300 MHz CDCl3 7.92 (s, 1H), 7.4 (s, 2H), 1.9 (m, 2H), 7.75 (s,1H) 7.22 (m, 1H), 7.05 (s, 1H), 2.28 (s, 1.56 (s, 3H), 6.5 (m, 1H) 6H)1.13 (t, 3H) RG-120112 300 MHz CDCl3 7.4 (m, 2H), 7.5 (2s, 1H), 1.7-2.2(m, 8H) 6.3 (s, 1H), 7.25 (m, 2H), 7.1 (s, 1H), 2.27 (s, 7.8 (s, 1H)7.15 (t, 1H) 6H) RG-120117 300 MHz CDCl3 7.35 (m, 3H), 7.65 (d, 2H), 1.6(s, 6H) 7.2 (m, 2H), 7.4 (d, 2H) 4.45 (s, 2H), 4.0 (s, 2H) RG-120120 300MHz CDCl3 7.23 (m, 3H), 7.21 (s, 2H), 1.8 (br, 2H), 6.9 (s, 1H) 7.05 (m,2H), 7.1 (s, 1H), 2.28 (s, 1.4 (br s, 3H), 3.45 (s, 2H) 6H) 1.05 (t, 3H)RG-120121 300 MHz CDCl3 7.3 (m, 3H), 7.55 (d, 2H), 1.47 (s, 6H) 6.9 (s,1H) 7.1 (m, 2H), 3.47 (s, 7.35 (d, 2H) 2H) RG-120124 300 MHz CDCl3 7.4(d, 2H), 7.8 (d, 2H), 3.98 (m, 2H), 7.2 (d, 2H), 2.65 (q, 7.55 (m, 3H)3.9 (m, 2H), 2H), 1.21 (t, 3H) 2.1 (br, 4H) RG-120125 300 MHz CDCl3 7.8(d, 2H), 7.6 (m, 3H), 2.1 (br, 4H), 7.23 (d, 2H), 2.65 (q, 7.4 (m, 2H)1.8 (br, 4H) 2H), 1.21 (t, 3H) RG-120130 300 MHz CDCl3 7.4 (m, 2H), 7.8(d, 1H), 3.8-4.0 (m, 4H), 7.25 (m, 2H), 7.65 (d, 2H), 7.5 (m, 1.85 (brm, 4H) 7.0 (m, 1H), 2H) 3.44 (s, 2H) RG-120132 300 MHz CDCl3 7.1-7.7 (m,10H) 1.8-2.3 (m, 8H) 7.85 (s, 1H), 6.25 (s, 1H) RG-120138 300 MHz CDCl37.4 (m, 1H), 7.4 (s, 2H), 3.95 (br, 2H), 7.8 (s, 1H) 7.2 (m, 1H), 6.5(m, 7.07 (s, 1H), 2.29 (s, 3.85 (m, 2H), 1H) 6H) 2.05 (m, 4H) RG-120141300 MHz CDCl3 7.27 (t, 2H), 7.32 (s, 2H), 1.8 (m, 2H), 7.87 (s, 1H) 7.0(t, 1H), 6.8 (d, 7.1 (s, 1H), 2.3 (s, 1.45 (s, 3H), 2H), 4.5 (s, 2H) 6H)0.9 (t, 3H) RG-120142 300 MHz CDCl3 7.9 (d, 2H), 7.45 (m, 2H), 1.66 (s,6H) 7.22 (d, 2H), 2.65 (q, 7.4 (m, 2H), 2H), 1.21 (t, 3H) 7.6 (m, 5H)RG-120150 300 MHz DMSO-d6 8.05 (br s, 1H), 7.7 (m, 2H), 2.15 (br, 2H),4.6 (s, 1H) 7.99 (d, 1H), 7.45 (m, 3H) 1.85 (br, 2H), 7.92 (d, 1H), 1.65(br s, 4H) 7.75 (d, 1H), 7.3 (m, 1H) RG-120152 300 MHz CDCl3 7.8 (m,3H), 7.45 (s, 2H), 2.05 (m, 4H), 7.40 (m, 2H) 7.05 (s, 1H), 1.85 (m, 4H)2.27 (s, 6H) RG-120155 300 MHz CDCl3 7.4 (m, 3H), 7.77 (d, 2H), 2.05 (brs, 4H), 7.95 (s, 1H) 7.15 (m, 2H), 7.3 (m, 3H) 1.8 (br s, 4H) 4.40 (s,2H), 4.0 (s, 2H) RG-120158 300 MHz CDCl3 7.77 (d, 2H), 7.42 (m, 3H),2.05 (br s, 4H), 7.95 (s, 1H) 7.02 (t, 1H), 7.3 (m, 2H) 1.8 (m, 4H) 6.8(d, 2H), 4.49 (s, 2H) RG-120159 300 MHz CDCl3 7.1 (t, 1H), 7.8 (m, 2H),4.0 (m, 2H), 7.22 (s, 1H) 6.9 (d, 1H), 6.55 (d, 7.5 (m, 3H) 3.9 (m, 2H),2.1 (m, 1H), 2.4 (br, 2H), 1.9 (m, 2H) 2H), 0.95 (t, 3H) RG-121517 500MHz CDCl3 6.55 (d, 1H), 7.8 (m, 2H), 1.62 (s, 6H) 6.45 (d, 1H), 7.45 (m,3H) 4.3 (m, 4H), 2.55 (m, 2H), 1.05 (t, 3H) RG-121518 500 MHz CDCl3 6.73(d, 1H), 7.31 (s, 1H), 2.33 (s, 6H), 7.4 (br s, 1H) 6.64 (d, 1H), 7.1(s, 2H) 1.8 (m, 2H), 1.49 (s, 4.3 (m, 4H), 2.6 (br, 3H), 1.03 (t, 3H)2H), 1.14 (t, 3H) RG-121513 500 MHz CDCl3 7.9 (t, 1H), 7.37 (s, 2H), 1.9(m, 2H), 7.2 (d, 1H), 6.95 (d, 7.0 (s, 1H), 2.3 (s, 1.57 (s, 3H), 1H),2.7 (q, 2H), 6H) 1.22 (t, 3H) 1.13 (t, 3H) RG-121514 500 MHz CDCl3 8.1(d, 1H), 7.8 (d, 2H), 2.1 (br s, 4H), 7.9 (t, 1H), 7.1 (d, 7.4 (m, 3H)1.9 (m, 2H), 1H), 6.95 (d, 1.8 (m, 2H) 1H), 2.65 (q, 2H), 1.2 (t, 3H)RG-121515 500 MHz CDCl3 8.0 (d, 1H 7.4 (s, 2H), 2.1 (br s, 4H), [NH]),7.9 (m, 7.05 (s, 1H), 2.28 (s, 1.87 (m, 2H), 1H), 7.1 (m, 6H) 1.75 (m,2H) 1H), 6.95 (d, 2H), 2.7 (q, 2H), 1.2 (t, 3H) RG-121516 500 MHz CDCl38.05 (d 1H 7.8 (m, 2H), 1.65 (s, 6H) [NH]), 7.9 (m, 7.4 (m, 3H) 1H), 7.1(d, 1H), 6.9 (d, 1H), 2.65 (q, 2H), 1.2 (t, 3H)

Example 2 Biological Testing of Compounds

The ligands of the present invention are useful in various applicationsincluding gene therapy, expression of proteins of interest in hostcells, production of transgenic organisms, and cell-based assays.

27-63 Assay Gene Expression Cassette

GAL4 DBD (1-147-CfEcR(DEF)/VP16AD-βRXREF-LmUSPEF: The wild-type D, E,and F domains from spruce budworm Choristoneura fumiferana EcR(“CfEcR-DEF”; SEQ ID NO: 1) were fused to a GAL4 DNA binding domain(“Gal4 DBD1-147”; SEQ ID NO: 2) and placed under the control of aphosphoglycerate kinase promoter (“PGK”; SEQ ID NO: 3). Helices 1through 8 of the EF domains from Homo sapiens RXRβ (“HsRXRβ-EF”;nucleotides 1465 of SEQ ID NO: 4) and helices 9 through 12 of the EFdomains of Locusta migratoria Ultraspiracle Protein (“LmUSP-EF”;nucleotides 403-630 of SEQ ID NO: 5) were fused to the transactivationdomain from VP16 (“VP16AD”; SEQ ID NO: 6) and placed under the controlof an elongation factor-1α promoter (“EF-1α”; SEQ ID NO: 7). Fiveconsensus GAL4 response element binding sites (“5XGAL4RE”; comprising 5copies of a GAL4RE comprising SEQ ID NO: 8) were fused to a syntheticTATA minimal promoter (SEQ ID NO: 9) and placed upstream of theluciferase reporter gene (SEQ ID NO: 10).

Stable Cell Line

CHO cells were transiently transfected with transcription cassettes forGAL4 DBD (1-147) CfEcR(DEF) and for VP16AD βRXREF-LmUSPEF controlled byubiquitously active cellular promoters (PGK and EF-1α, respectively) ona single plasmid. Stably transfected cells were selected by Zeocinresistance. Individually isolated CHO cell clones were transientlytransfected with a GAL4 RE-luciferase reporter (pFR Luc). 27-63 clonewas selected using Hygromycin.

Treatment with Ligand

Cells were trypsinized and diluted to a concentration of 2.5×10⁴ cellsmL. 100 μL of cell suspension was placed in each well of a 96 well plateand incubated at 37° C. under 5% CO₂ for 24 h. Ligand stock solutionswere prepared in DMSO and diluted 300 fold for all treatments. Doseresponse testing consisted of 8 concentrations ranging from 33 μM to0.01 μM.

Reporter Gene Assay

Luciferase reporter gene expression was measured 48 h after celltreatment using Bright-Glo™ Luciferase Assay System from Promega(E2650). Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

Z3 Assay Stable Cell Line

Dr. F. Gage provided a population of stably transformed cells containingCVBE and 6XEcRE as described in Suhr, S. T., Gil, E. B., Senut M. C.,Gage, F. H. (1998) Proc. Natl. Acad. Sci. USA 95, 7999-804. Human 293kidney cells, also referred to as HEK-293 cells, were sequentiallyinfected with retroviral vectors encoding first the switch constructCVBE, and subsequently the reporter construct 6XEcRE Lac Z. The switchconstruct contained the coding sequence for amino acids 26-546 fromBombyx mori EcR (BE) (Iatrou) inserted in frame and downstream of theVP16 transactivation domain (VBE). A synthetic ATG start codon wasplaced under the control of cytomegalovirus (CVBE) immediate earlypromoter and flanked by long terminal repeats (LTR). The reporterconstruct contained six copies of the ecdysone response element (EcRE)binding site placed upstream of LacZ and flanked on both sides with LTRsequences (6XEcRE).

Dilution cloning was used to isolate individual clones. Clones wereselected using 450 ug/mL G418 and 100 ng/mL puromycin. Individual cloneswere evaluated based on their response in the presence and absence oftest ligands. Clone Z3 was selected for screening and SAR purposes.

Human 293 kidney cells stably transformed with CVBE and 6XEcRE LacZ weremaintained in Minimum Essential Medium (Mediates, 10-010-CV) containing10% FBS (Life Technologies, 26140-087), 450 gum G418 (Mediates,30-234-CR), and 100 gnome promising (Sigma, P-7255), at 37° C. in anatmosphere containing 5% CO₂ and were subculture when they reached 75%confluence.

Treatment with Ligand

Z3 cells were seeded into 96-well tissue culture plates at aconcentration of 2.5×10³ cells per well and incubated at 37° C. in 5%CO₂ for twenty-four hours. Stock solutions of ligands were prepared inDMSO. Ligand stock solutions were diluted 100 fold in media and 50 μL ofthis diluted ligand solution (33 μM) was added to cells. The finalconcentration of DMSO was maintained at 0.03% in both controls andtreatments.

Reporter Gene Assays

Reporter gene expression was evaluated 48 hours after treatment ofcells, β-galactosidase activity was measured using Gal Screen™bioluminescent reporter gene assay system from Tropix (GSY1000). Foldinduction activities were calculated by dividing relative light units(“RLU”) in ligand treated cells with RLU in DMSO treated cells.Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

A schematic of switch construct CVBE, and the reporter construct 6XEcRELac Z is shown in FIG. 1. Flanking both constructs are long terminalrepeats, G418 and puromycin are selectable markers, CMV is thecytomegalovirus promoter, VBE is coding sequence for amino acids 26-546from Bombyx mori EcR inserted downstream of the VP16 transactivationdomain, 6×EcRE is six copies of the ecdysone response element, lacZencodes for the reporter enzyme β-galactosidase,

13B3 Assay Gene Expression Cassette

GAL4 DBD-CfEcR(DEF)/VP16AD-MmRXRE: The wild-type D, E, and F domainsfrom spruce budworm Choristoneura fumiferana EcR (“CfEcR-DEF”; SEQ IDNO: 1) were fused to a GAL4 DNA binding domain (“Gal4DBD1-147”; SEQ EDNO: 2) and placed under the control of the SV40e promoter of pM vector(PIT3119-5, Clontech, Palo Alto, Calif.). The D and E domains from MusMusculus RXR (“MmRXR-DE”; SEQ ID NO: 11) were fused to thetransactivation domain from VP16 (“VP16AD”; SEQ ID NO: 6) and placedunder the control of the SV40e promoter of the pVP16 vector (PT3127-5,Clontech, Palo Alto, Calif.).

Stable Cell Line

CHO cells were transiently transfected with transcription cassettes forGAL4 DBD-CfEcR(DEF) and for VP16AD-M mR controlled by SV40e promoters.Stably transfected cells were selected using Hygromycin. Individuallyisolated CHO cell clones were transiently transfected with a GAL4RE-luciferase reporter (pFR-Luc, Stratagene, La Jolla, Calif.). The 13B3clone was selected using Zeocin.

Treatment with Ligand

Cells were trypsinized and diluted to a concentration of 2.5×10⁴ cellsmL. 100 μL of cell suspension was placed in each well of a 96 well plateand incubated at 37° C. under 5% CO₂ for 24 h. Ligand stock solutionswere prepared in DMSO and diluted 300 fold for all treatments. Doseresponse testing consisted of 8 concentrations ranging from 33 μM to0.01 μM.

Reporter Gene Assay

Luciferase reporter gene expression was measured 48 h after celltreatment using Bright-Glo™ Luciferase Assay System from Promega(E2650). Luminescence was detected at room temperature using a Dynex MLXmicrotiter plate luminometer.

The results of the assays are shown in Tables 5 and 6. Each assay wasconducted in two separate wells, and the two values were averaged. Foldinductions were calculated by dividing relative light units (“RLU”) inligand treated cells with RLU in DMSO treated cells. EC₅₀s werecalculated from dose response data using a three-parameter logisticmodel. Relative Max Ft was determined as the maximum fold induction ofthe tested ligand (an embodiment of the invention) observed at anyconcentration relative to the maximum fold induction of GS-™-E ligand(3,5-Dimethyl-benzoic acidN-tert-butyl-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide) observed at anyconcentration.

TABLE 5 Biological Assay Results for Compounds: Fold Induction FoldInduction Average 13B3 assay 27-63 assay Z3 assay Compound 33/33.3 (μM)33.3 (μM) 33 (μM) RG-103441 1.6 0.8 RG-103468 2.2 1.1 RG-120001 3.3 0.3RG-120002 0.2 5.7 3.9 RG-120005 0.8 RG-120006 1.1 0.5 RG-120008 0.0567.3 RG-120009 1.3 0.8 RG-120012 0.7 0.7 RG-120014 0.0 1.1 RG-1200150.0 0.8 RG-120016 4.4 1146.6 226.9 RG-120017 0.8 0.9 RG-120018 0.8 0.7RG-120019 1.5 RG-120020 1.3 0.2 RG-120021 0.0 0.7 RG-120022 0.0 0.3 0.1RG-120023 0.0 1.4 0.2 RG-120024 2.1 6.3 3.8 RG-120025 0.9 0.8 RG-1200260.9 8.5 41.1 RG-120029 1.5 0.7 RG-120033 0.9 0.7 RG-120035 1.2 9.1RG-120037 0.3 0.4 0.5 RG-120038 0.0 0.2 RG-120040 174.4 241.6 202.3RG-120042 3.3 19.3 RG-120045 1412.3 2707.3 275.8 RG-120046 1.3 8.9RG-120047 973.9 31.9 RG-120048 0.0 1.0 RG-120049 2661.5 2070.7 310.0RG-120050 0.9 3.5 RG-120052 0.7 4.9 RG-120055 0.7 0.5 0.2 RG-120056 0.11.0 RG-120057 0.6 0.8 RG-120058 0.3 0.3 1.7 RG-120059 0.0 0.6 RG-12006016.8 108.5 RG-120061 0.4 0.9 0.6 RG-120062 0.2 0.6 RG-120066 1.4 0.6RG-120067 2.6 0.5 RG-120069 477.7 1872.1 RG-120070 0.4 0.7 RG-120072 0.61.3 RG-120073 0.9 0.5 RG-120075 1.1 0.7 RG-120076 20.2 4091.3 0.2RG-120077 0.4 14.6 RG-120078 0.0 0.4 RG-120079 0.7 0.6 RG-120080 716.9310.4 RG-120081 0.5 0.6 RG-120082 0.1 0.8 RG-120083 1.3 0.8 RG-1200864.0 111.6 RG-120087 0.4 0.4 RG-120088 0.9 8.2 RG-120091 0.5 1.5RG-120092 2186.5 RG-120093 0.0 5.0 0.2 RG-120094 0.6 0.8 RG-120096 0.20.7 RG-120098 0.3 1.8 RG-120099 0.0 1327.0 0.2 RG-120101 1.2 0.7RG-120102 0.5 0.8 RG-120105 0.5 0.8 RG-120108 0.0 0.3 RG-120109 0.0 0.2RG-120110 0.8 0.5 RG-120111 0.6 0.7 RG-120113 3.4 0.6 RG-120114 1.1 0.6RG-120115 0.8 9.1 RG-120117 1.2 0.8 RG-120118 0.8 13.4 1.4 RG-120119 0.40.8 RG-120121 0.1 0.7 RG-120122 0.5 0.6 RG-120124 1.2 1.0 2.0 RG-12012582.6 509.9 253.6 RG-120126 0.4 2.4 RG-120127 0.6 0.7 RG-120128 129.0338.2 403.0 RG-120129 0.9 0.4 RG-120134 0.3 0.6 RG-120135 0.4 0.7 0.7RG-120136 0.3 0.6 RG-120137 5.3 4.6 59.0 RG-120140 1.4 0.5 RG-120142 0.30.4 RG-120144 0.1 0.7 RG-120145 1.4 0.5 RG-120147 1.1 1.0 RG-120148 0.00.7 1.1 RG-120149 1.1 0.7 RG-120151 0.3 0.8 RG-120152 264.3 59.9RG-120153 0.8 0.7 RG-120156 1.4 0.7 RG-120157 0.0 0.9 RG-120159 0.1 3.70.2 RG-120160 0.3 0.6 RG-120161 59.9 RG-120162 0.9 0.7 RG-120163 1.1 0.5RG-120164 1.7 0.6 RG-120326 0.0 RG-121513 1015.2 RG-121514 1.7 RG-12151536.6 RG-121516 7.9 RG-121517 3514.1 RG-121518 2336.5

TABLE 6 Biological Assay Results for Compounds: EC50/Relative Max FIEC50 (μM)/Rel EC50 (μM)/Rel EC50 (μM)/Rel Max FI Max FI Max FI Compound13B3 assay 27-63 assay Z3 assay RG-120002 >33/0 RG-120005 >33/0RG-120016   ~20/0.71 RG-120019 >33/0 RG-120022 >33/0 RG-120023 >33/0RG-120024 >33/0 RG-120026 >33/0 RG-120037 >33/0 RG-120040   ~20/0.13RG-120042 >33/0 RG-120045   ~20/1.25 RG-120049 3.57/1.56  3.42/1.58 1.6/0.88 RG-120055 >33/0 RG-120058 >33/0 RG-120060   >33/0.01RG-120061 >33/0 RG-120069 2.95/1.02  3.31/1.11 1.68/0.8 RG-120076 >33.3/0.05    ~20/1.4 12.43/0.9  RG-120080 12.35/1.04  8.35/1.29 3.95/0.71 RG-120092  7.16/1.15 RG-120093 >33/0RG-120096 >33.3/0    >50/0  RG-120099   ~20/0.64 RG-120115 >33/0RG-120118 >33/0 RG-120124 >33/0 RG-120125 >33.3/0.16    ~20/0.2710.02/0.67  RG-120126 >33.3/0     >50/0.04 RG-120128   ~20/0.18RG-120135 >33/0 RG-120137 >33/0 RG-120148 >33/0 RG-120159 >33/0RG-120161 3.46/0.14 2.14/0.08 RG-121513   ~10/0.44 RG-121514  3.89/0.5RG-121515    ~5/0.17 RG-121516 >33/0 RG-121517   ~20/1.55 RG-121518 3.57/1.08

Example 3 Insecticidal Activity of Compounds

The compound to be evaluated was dissolved in an appropriate solvent,usually a mix of acetone, methanol, and water. Test solutions were madeby serial dilution of a stock test solution with acetone, methanol, andwater. Initial evaluations were made at one or more concentrations onone or more of the following insects:

Code Symbol Common Name Latin Name BAW Beet Armyworm Spodoptera exiguaCL Cabbage Looper Trichoplusia ni TBW Tobacco Budworm Heliothisvirescens

Feeding bioassays were conducted in bioassay trays containing insectdiet. Treatments were made by applying 50 mL of test solution to thesurface of the diet in each of 5 wells. After the test solution dried,each well was infested with a single neonate larva. The trays were heldfor six days and then the mortality rating was determined for eachtreatment.

Contact bioassays (green peach aphid, two-spotted spider mite, whitefly) were conducted by applying a solution of the test compound to theinside surface of a Petri dish. The solution was allowed to air-dry,then each dish was infested and the larvae from each treatment weretransferred to a bioassay tray. The trays were held for one to sevendays, and the mortality rating was determined for each treatment.

Some of the tobacco budworm tests were conducted as follows. A testsolution containing 600 ppm was made by dissolving a compound of theinvention in a 1:1 acetone:methanol solution, then adding water to givea 5:5:90 acetone:methanol:water solution, and finally a surfactant wasadded at an equivalent of 7.37 g of surfactant per 100 L of testsolution (1 ounce of surfactant per 100 gallons of test solution).Appropriate dilutions were prepared in water from the 600 ppm solution.A detached cotton leaf, Gossypium hirsutum was placed on moistenedfilter paper in a Petri dish (100×20 mms). The leaf was sprayed with thetest solution using a rotating turntable sprayer and allowed to dry. Thedish was infested with 10 first instar larvae of the tobacco budworm andcovered with the lid. If the larvae were alive two days after treatment,fresh untreated cotton leaves were added. All treatments were maintainedat 23.9-26.7° C. (75-80° F.) under fluorescent light in awell-ventilated room. Percent mortality was determined at four daysafter treatment.

TABLE 7 Insecticidal Activity Assay Results for Compounds CfEcR(CDEF)/CfUSP, GST fusion protein Insect Toxicity (LC50 (ppm) or %control @ 150 ppm) Compound CfEcR EC50 MTA BAW CL TBW WFN RG-120096 6% @10 uM  74 @ 150  0% @ 150 60% @ 150  0% @ 150 55% @ 150  RG-120076 8.35nM 92 47 47 >150 >150 RG-120128 28.5 nM (28% @ 1 uM) >150 4717 >150 >150 RG-120039 203 nM (21% @ 1 uM) RG-120126 136 nM 0% @ 150  0%@ 150 0% @ 150 0% @ 150 79% @ 150  RG-120008 81% @ 1 uM RG-120080 Kd =11.9 nM 0% @ 150 23 47 >150 0% @ 150 RG-120060 Kd = 38 nM 0% @ 150 2347 >150 0% @ 150 RG-120161 62.4 nM 110 87 17 >150 >150 RG-120051 147 nMRG-120069 14.2 nM 146 4.7 4.7 >150 >150 RG-120125 57.9 nM 167 98 23 >150150 RG-120092 99% @ 1 uM >150 47 13 98 >150 RG-120152 92% @ 1 uMRG-120035 0% @ 150 40% @ 150 100% @ 150  0% @ 150 0% @ 150 RG-120099 0%@ 150 100% @ 150  40% @ 150  0% @ 150 0% @ 150 RG-120015 0% @ 150 80% @150 0% @ 150 0% @ 150 0% @ 150 RG-120001 0% @ 150 80% @ 150 20% @ 150 0% @ 150 0% @ 150 RG-120021 51% @ 150  100% @ 150  0% @ 150 0% @ 150 0%@ 150 RG-120026 40% @ 150  100% @ 150  0% @ 150 0% @ 150 0% @ 150RG-120042 481 nM (Plodia EC50); 29 uM (Kc EC50) RG-120115 inactive asinsecticide RG-120077 0% @ 150 50% @ 150 100% @ 150  0% @ 150 0% @ 150

In addition, one of ordinary skill in the art is also able to predictthat the ligands disclosed herein will also work to modulate geneexpression in various cell types described above using gene expressionsystems based on group H and group B nuclear receptors.

1.-5. (canceled)
 6. A method of modulating the expression of a targetgene in a host cell, wherein the host cell includes a first geneexpression cassette comprising a first polynucleotide encoding a firstpolypeptide comprising: (i) a transactivation domain; (ii) a DNA-bindingdomain; and (iii) a Group H nuclear receptor ligand binding domain; asecond gene expression cassette comprising: (i) a response elementcapable of binding to said DNA binding domain; (ii) a promoter that isactivated by the transactivation domain; and (iii) said target gene; themethod comprising contacting said host cell with a compound of theformula:

wherein X and X′ are independently O or S; R¹ is a) H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, or benzyloxy; b) unsubstituted or substituted phenylwherein the substituents are independently 1 to 5H; halo; nitro; cyano;hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁-C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a));carboxamido(CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(b)R^(c));mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a 5- or6-membered dioxolano or dioxano heterocyclic ring; c) unsubstituted orsubstituted naphthyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; f) unsubstituted orsubstituted benzothiophene-2-yl, benzothiophene-3-yl, benzofuran-2-yl,or benzofuran-3-yl wherein the substituents are independently 1 to 3halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)); e) unsubstituted or substituted 2, 3,or 4-pyridyl wherein the substituents are independently 1 to 3 halo,cyano, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or(C₁-C₆)haloalkoxy; f) unsubstituted or substituted 5-memberedheterocycle selected from furyl, thiophenyl, triazolyl, pyrrolyl,isopyrroyl, pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl, oxazolyl,or isooxazolyl wherein the substituents are independently 1 to 3 halo,nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,carboxy, (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)), or amino (—NR^(a)R^(b)); g)aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein thearomatic substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or h) aromatic-substituted orunsubstituted phenylamino, phenyl(C₁-C₆)alkylamino, orphenylcarbonylamino wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; whereinR^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl, or phenyl; R²and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)_(x)—), analkyloxylalkyl linkage (—(CH₂)_(y)—O(CH₂)_(z)—), an alkylaminoalkyllinkage (—CH₂)_(y)NR^(a)(CH₂)_(z)—), or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon atom towhich they are attached, wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a)is H, (C₁-C₆)alkyl, or phenyl; and R⁴ is unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5H; halo; nitro;cyano; hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁-C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(b)R^(c));mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined to form a 5- or 6-membered dioxolano (—OCH₂O—) or dioxano(—OCH₂CH₂O—) heterocyclic ring; wherein R^(a), R^(b), and R^(c) areindependently H, (C₁-C₆)alkyl, or phenyl; provided that R⁴ is not3-nitrophenyl or 4-nitrophenyl, and when R⁴ is phenyl, then R¹ is notphenyl, when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, or whenR⁴ is 4-chlorophenyl, then R¹ is not methyl.
 7. The method of claim 6wherein the compound is of the specified formula and: X and X′ areindependently O or S; R¹ is a) H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₆)alkoxy, orbenzyloxy; b) unsubstituted or substituted phenyl wherein thesubstituents are independently 1 to 5H; halo; nitro; cyano; hydroxy;(C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl;(C₁-C₆)alkoxy; (C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄) alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); (C₁-C₆)alkylsulfonyl;(C₁-C₆)alkylsulfoxido (—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); orunsubstituted or substituted phenyl wherein the substituents areindependently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino;or when two adjacent positions on the phenyl ring are substituted withalkoxy groups, these groups, together with the carbon atoms to whichthey are attached, may be joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—)to form a 5- or 6-membered dioxolano or dioxano heterocyclic ring; c)unsubstituted or substituted benzothiophene-2-yl, or benzofuran-2-ylwherein the substituents are independently 1 to 3 halo, nitro, hydroxy,(C₁-C₆)alkyl, or (C₁-C₆)alkoxy; d) unsubstituted or substituted 2, 3, or4-pyridyl wherein the substituents are independently 1 to 3 halo, cyano,nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or (C₁-C₆)haloalkoxy; e)unsubstituted or substituted 5-membered heterocycle selected from furyl,thiophenyl, triazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, orisooxazolyl wherein the substituents are independently 1 to 3 halo,nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,carboxy, or (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)); f) aromatic-substitutedor unsubstituted phenyl(C₁-C₆)alkyl, phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, orphenoxy(C₁-C₆)alkyl wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, or (C₁-C₆)alkyl; or g)aromatic-substituted or unsubstituted phenylamino,phenyl(C₁-C₆)alkylamino, or phenylcarbonylamino wherein the aromaticsubstituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, or(C₁-C₆)alkyl; wherein R^(a) and R^(b) are independently H, (C₁-C₆)alkyl,or phenyl; R² and R³ are independently H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl,(C₁-C₆)alkoxy(C₁-C₆)alkyl, phenyl, or together as an alkane linkage(—(CH₂)_(x)—), an alkyloxylalkyl linkage (—(CH₂)_(y)—O—(CH₂)_(z)—), analkylaminoalkyl linkage (—(CH₂)_(y)NR^(a)(CH₂)_(x)—), or analkylbenzoalkyl linkage (—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ringwith the carbon atom to which they are attached, wherein x=3 to 7, y=1to 3, z=1 to 3, and R^(a) is H, (C₁-C₆)alkyl, or phenyl; and R⁴ isunsubstituted or substituted phenyl wherein the substituents areindependently 1 to 5H; halo; nitro; cyano; hydroxy; (C₁-C₆)alkyl;(C₁-C₆)haloalkyl; (C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄) alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁-C₆)alkoxycarbonyl; (C₁-C₆)alkanoyloxy(—OCOR^(a));carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b));(C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido (—S(O)R^(a));sulfamido(—SO₂NR^(a)R^(b)); or unsubstituted or substituted phenylwherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacent positions onthe phenyl ring are substituted with alkoxy groups, these groups,together with the carbon atoms to which they are attached, may be joinedas a linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a 5- or 6-membereddioxolano or dioxano heterocyclic ring; wherein R^(a) and R^(b) areindependently H, (C₁-C₆)alkyl, or phenyl; provided that R⁴ is not3-nitrophenyl or 4-nitrophenyl, and when R⁴ is phenyl, then R¹ is notphenyl, when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, or whenR⁴ is 4-chlorophenyl, then R¹ is not methyl.
 8. The method of claim 7wherein the compound is of the specified formula and: X is O; X′ is O orS; R¹ is a) H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl, or(C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl; b) unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5H; halo; nitro;cyano; (C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)alkoxy; (C₁-C₆)haloalkoxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)alkoxycarbonyl; carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); or phenyl; or when twoadjacent positions on the phenyl ring are substituted with alkoxygroups, these groups, together with the carbon atoms to which they areattached, may be joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a5- or 6-membered dioxolano or dioxano heterocyclic ring; c)unsubstituted or substituted benzothiophene-2-yl, or benzofuran-2-ylwherein the substituents are independently 1 to 3 halo, nitro, hydroxy,(C₁-C₆)alkyl, or (C₁-C₆)alkoxy; d) unsubstituted or substituted furyl orthiophenyl wherein the substituents are independently 1 to 3 halo,nitro, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, (C₁-C₆)alkoxycarbonyl(—CO₂R^(a)), or phenyl; e) aromatic-substituted or unsubstitutedphenyl(C₁-C₆)alkyl, phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, orphenoxy(C₁-C₆)alkyl wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, or (C₁-C₆)alkyl; or f)aromatic-substituted or unsubstituted phenylamino,phenyl(C₁-C₆)alkylamino, or phenylcarbonylamino wherein the aromaticsubstituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, or(C₁-C₆)alkyl; wherein R^(a) and R^(b) are independently H, (C₁-C₆)alkyl,or phenyl; R² and R³ are independently H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl, phenyl, or together as analkane linkage (—(CH₂)_(x)—), an alkyloxylalkyl linkage(—(CH₂)_(y)—O—(CH₂)_(z)—), an alkylaminoalkyl linkage(—(CH₂)_(y)NR_(a)(CH₂)_(z)—), or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon atom towhich they are attached, wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a)is H, (C₁-C₆)alkyl, or phenyl; and R⁴ is unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5H; halo; nitro;cyano; (C₁-C₆)alkyl; (C₁-C₆)haloalkyl; (C₁-C₆)alkoxy; (C₁-C₆)haloalkoxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)alkoxycarbonyl; carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); or phenyl; or when twoadjacent positions on the phenyl ring are substituted with alkoxygroups, these groups, together with the carbon atoms to which they areattached, may be joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a5- or 6-membered dioxolano or dioxano heterocyclic ring; wherein R^(a)and R^(b) are independently H, (C₁-C₆)alkyl, or phenyl; provided that R⁴is not 3-nitrophenyl or 4-nitrophenyl, and when R⁴ is phenyl, then R¹ isnot phenyl, when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, orwhen R⁴ is 4-chlorophenyl, then R¹ is not methyl.
 9. The method of claim8 wherein the compound is of the specified formula and: X and X′ are O;R¹ is phenyl, 4-chlorophenyl-, 4-ethylphenyl-,2-ethyl-3,4-ethylenedioxyphenyl, 3-fluorophenyl-,2-fluoro-4-ethylphenyl-, 2-methyl-3-methoxyphenyl-,2-ethyl-3-methoxyphenyl, 3-methylphenyl-, 2-methoxyphenyl-,2-nitrophenyl-, 3-nitrophenyl-, 2-furanyl-, benzyl-,benzothiophene-2-yl-, phenylamino-, benzyloxymethyl, phenoxymethyl-,3-toluoylamino-, benzylamino-, benzoylamino-, ethoxycarbonylethyl-, or3-chloro-2,2,3,3-tetrafluoroethyl; R² and R³ are independently methyl,ethyl, or together as a tetramethylene (—(CH2)₄—), 4-pyrano(—CH₂CH₂OCH₂CH₂—), or methylenebenzoethylene (—CH₂-1-benzo-2-CH₂CH₂—)linkage form a ring with the carbon atom to which they are attached; andR⁴ is phenyl, 4-biphenyl, 4-chlorophenyl, 2,4-dimethoxyphenyl,3,5-dimethylphenyl, 2-methoxyphenyl, 3,4-methylenedioxyphenyl,3-trifluoromethylphenyl, or 4-trifluoromethoxyphenyl; provided that whenR⁴ is phenyl, then R¹ is not phenyl.
 10. The method of claim 9, whereinthe compound is selected from the group consisting of:1-Benzyl-3-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-urea;1-Benzoyl-3-[3-(3,5-dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-urea;N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;3-Chloro-N-[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2,2,3,3-tetrafluoropropionamide;N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;Benzo[b]thiophene-2-carboxylic acid[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-succinamicacid ethyl ester;1-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;2-Benzyloxy-N-[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-acetamide;Furan-2-carboxylic acid[3-(4-chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;N-[3-(4-Chloro-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;N-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-benzamide;N-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;Benzo[b]thiophene-2-carboxylic acid[5,5-dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-amide;1-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;N-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;2-Benzyloxy-N-[5,5-dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-acetamide;N-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;Furan-2-carboxylic acid[5,5-dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]amide;N-[5,5-Dimethyl-3-(4-trifluoromethoxy-phenyl)-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxybenzamide;N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-benzamide;3-Chloro-N-[5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2,2,3,3-tetrafluoro-propionamide;N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-succinamicacid ethyl ester;1-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;2-Benzyloxy-N-[5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-acetamide;Furan-2-carboxylic acid[5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]amide;4-Ethyl-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxy-benzamide;N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;N-[5,5-Dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;Benzo[b]thiophene-2-carboxylic acid[5,5-dimethyl-3-(3-trifluoromethyl-phenyl)-[1,2,4]oxadiazol-4-yl]-amide;3-Chloro-2,2,3,3-tetrafluoro-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-propionamide;N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-succinamicacid ethyl ester; Benzo[b]thiophene-2-carboxylic acid[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;1-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;2-Benzyloxy-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-acetamide;N-[3-(2-Methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;Furan-2-carboxylic acid[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;2-Ethyl-3-methoxy-N-[3-(2-methoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-benzamide;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-benzamide;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-succinamicacid ethyl ester; Benzo[b]thiophene-2-carboxylic acid(3-benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-amide;1-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-3-phenyl-urea;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-phenoxy-acetamide;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-benzyloxy-acetamide;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-phenyl-acetamide;Furan-2-carboxylic acid(3-benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-amide;N-(3-Benzo[1,3]dioxol-5-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-ethyl-3-methoxy-benzamide;N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-benzamide;N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-succinamicacid ethyl ester; Benzo[b]thiophene-2-carboxylic acid[3-(2,4-dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;1-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;2-Benzyloxy-N-[3-(2,4-dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-acetamide;N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;Furan-2-carboxylic acid[3-(2,4-dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-amide;N-[3-(2,4-Dimethoxy-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxybenzamide;N-(3-Biphenyl-4-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-benzamide;N-(3-Biphenyl-4-yl-5,5-dimethyl-[1,2,4]oxadiazol-4-yl)-2-ethyl-3-methoxy-benzamide;4-Ethyl-N-(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-benzamide;N-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-benzamide;Benzo[b]thiophene-2-carboxylic acid(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide;1-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-phenyl-urea;N-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-2-phenoxy-acetamide;2-Benzyloxy-N-(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-acetamide;N-(5-Ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-2-phenyl-acetamide;Furan-2-carboxylic acid(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide;2-Ethyl-N-(5-ethyl-5-methyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-3-methoxy-benzamide;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-benzamide;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-benzamide;3-Chloro-N-[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2,2,3,3-tetrafluoro-propionamide;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-succinamicacid ethyl ester; Benzo[b]thiophene-2-carboxylic acid[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-amide;1-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-3-phenyl-urea;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2-phenoxy-acetamide;2-Benzyloxy-N-[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-acetamide;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2-phenyl-acetamide;Furan-2-carboxylic acid[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]amide;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-2-ethyl-3-methoxybenzamide;4-Ethyl-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-benzamide;N-(3-Phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-benzamide;3-Chloro-2,2,3,3-tetrafluoro-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4.4]non-2-en-4-yl)-propionamide;N-(3-Phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-succinamic acidethyl ester; Benzo[b]thiophene-2-carboxylic acid(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)amide;1-Phenyl-3-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-urea;2-Phenoxy-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-acetamide;2-Benzyloxy-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-acetamide;2-Phenyl-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-acetamide;Furan-2-carboxylic acid(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-amide;2-Ethyl-3-methoxy-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-benzamide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-4-ethyl-benzamide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-benzamide;3-Chloro-N-[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-2,2,3,3-tetrafluoro-propionamide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-succinamicacid ethyl ester; Benzo[b]thiophene-2-carboxylic acid[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-amide;1-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-3-phenyl-urea;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-2-phenoxy-acetamide;2-Benzyloxy-N-[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-acetamide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-2-phenyl-acetamide;Furan-2-carboxylic acid[3-(3,5-dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-amide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-2-ethyl-3-methoxybenzamide;4-Ethyl-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-benzamide;N-(3-Phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-benzamide;1-Phenyl-3-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-urea;2-Phenoxy-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-acetamide;2-Benzyloxy-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-acetamide;2-Phenyl-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-acetamide;2-Ethyl-3-methoxy-N-(3-phenyl-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl)-benzamide;N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-4-ethyl-benzamide;N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-benzamide;1-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-3-phenyl-urea;N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-2-phenoxyacetamide;2-Benzyloxy-N-[3-(3,5-dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]acetamide;N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-2-phenylacetamide;Furan-2-carboxylic acid[3-(3,5-dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-amide;N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-2-ethyl-3-methoxybenzamide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,5]-7,8-benzo-dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide;N-[3-(3,5-Dimethyl-phenyl)-1,8-dioxa-2,4-diaza-spiro[4,5]dec-2-en-4-yl]-3-methoxy-2-methyl-benzamide;N-[3-(3,5-Dimethyl-phenyl)-5,5-dimethyl-[1,2,4]oxadiazol-4-yl]-3-methoxy-2-methyl-benzamide;N-[3-(3,5-Dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-4-ethyl-2-fluoro-benzamide;4-Ethyl-2-fluoro-N-(3-phenyl-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl)-benzamide;N-[3-(3,5-Dimethyl-phenyl)-1-oxa-2,4-diaza-spiro[4,4]non-2-en-4-yl]-4-ethyl-2-fluorobenzamide;N-(5,5-Dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-4-ethyl-2-fluoro-benzamide;5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid(5,5-dimethyl-3-phenyl-[1,2,4]oxadiazol-4-yl)-amide; and5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid[3-(3,5-dimethyl-phenyl)-5-ethyl-5-methyl-[1,2,4]oxadiazol-4-yl]-amide.11. A method to modulate the expression of one or more exogenous genesin a subject, comprising administering to the subject an effectiveamount of a ligand of the formula:

wherein X and X′ are independently O or S; R¹ is a) H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, or benzyloxy; b) unsubstituted or substituted phenylwherein the substituents are independently 1 to 5H; halo; nitro; cyano;hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁-C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a));carboxamido(CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(t)R′);mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O—) to form a 5- or6-membered dioxolano or dioxano heterocyclic ring; c) unsubstituted orsubstituted naphthyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; d) unsubstituted orsubstituted benzothiophene-2-yl, benzothiophene-3-yl, benzofuran-2-yl,or benzofuran-3-yl wherein the substituents are independently 1 to 3halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)); e) unsubstituted or substituted 2, 3,or 4-pyridyl wherein the substituents are independently 1 to 3 halo,cyano, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or(C₁-C₆)haloalkoxy; f) unsubstituted or substituted 5-memberedheterocycle selected from furyl, thiophenyl, thazolyl, pyrrolyl,isopyrrolyl, pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl,oxazolyl, or isooxazolyl wherein the substituents are independently 1 to3 halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁C₆)alkoxy, (C₁-C₆)haloalkoxy,carboxy, (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)), or amino (—NR^(a)R^(b)); g)aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein thearomatic substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or h) aromatic-substituted orunsubstituted phenylamino, phenyl(C₁-C₆)alkylamino, orphenylcarbonylamino wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; whereinR^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl, or phenyl; R²and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)₂—)_(x)—) analkyloxylalkyl linkage (—(CH₂)_(y)—O—(CH₂)_(z)—), an alkylaminoalkyllinkage (—(CH₂),NR^(a)(CH₂)_(z)—, or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon atom towhich they are attached, wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a)is H, (C₁-C₆)alkyl, or phenyl; and R⁴ is unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5H; halo; nitro;cyano; hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl; (C₁C₆)alkoxy(C₁-C₆)alkoxy;(C₁-C₆)alkanoyloxy(C₁-C₆)alkyl; (C₂-C₆)alkenyl optionally substitutedwith halo, cyano, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy; (C₂-C₆)alkynyloptionally substituted with halo or (C₁-C₄)alkyl; formyl; carboxy;(C₁-C₆)alkylcarbonyl; (C₁-C₆)haloalkylcarbonyl; benzoyl;(C₁C₆)alkoxycarbonyl; (C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy(—OCOR^(a)); carboxamido(CONR^(a)R^(b)); amido (—NR^(a)COR^(b));alkoxycarbonylamino (—NR^(z)CO₂R^(b));alkylaminocarbonylamino(NR^(a)CONR^(b)R^(c)); mercapto;(C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined to form a 5- or 6-membered dioxolano (—OCH₂O—) or dioxano(—OCH₂CH₂O—) heterocyclic ring; wherein R^(a), R^(b), and R^(c) areindependently H, (C₁-C₆)alkyl, or phenyl; provided that R⁴ is not3-nitrophenyl or 4-nitrophenyl, and when R⁴ is phenyl, then R¹ is notphenyl, when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, or whenR⁴ is 4-chlorophenyl, then R¹ is not methyl.
 12. A method for regulatingendogenous or heterologous gene expression in a transgenic subjectcomprising contacting a ligand with an ecdysone receptor complex withinthe cells of the subject, wherein the cells further contain a DNAbinding sequence for the ecdysone receptor complex when in combinationwith the ligand and wherein formation of an ecdysone receptorcomplex-ligand-DNA binding sequence complex induces expression of thegene, and where the ligand has the following formula:

wherein X and X′ are independently O or S; R¹ is a) H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, or benzyloxy; b) unsubstituted or substituted phenylwherein the substituents are independently 1 to 5H; halo; nitro; cyano;hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a));carboxamido(CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(b)R^(c));mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined as a linkage (—OCH₂O—) or (—OCH₂CH₂O) to form a 5- or6-membered dioxolano or dioxano heterocyclic ring; c) unsubstituted orsubstituted naphthyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; d) unsubstituted orsubstituted benzothiophene-2-yl, benzothiophene-3-yl, benzofuran-2-yl,or benzofuran-3-yl wherein the substituents are independently 1 to 3halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)); e) unsubstituted or substituted 2, 3,or 4-pyridyl wherein the substituents are independently 1 to 3 halo,cyano, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or(C₁-C₆)haloalkoxy; f) unsubstituted or substituted 5-memberedheterocycle selected from furyl, thiophenyl, triazolyl, pyrrolyl,isopyrrolyl, pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl,oxazolyl, or isooxazolyl wherein the substituents are independently 1 to3 halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,carboxy, (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)), or amino (—NR^(a)R^(b)); g)aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein thearomatic substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or h) aromatic-substituted orunsubstituted phenylamino, phenyl(C₁-C₆)alkylamino, orphenylcarbonylamino wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; whereinR^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl, or phenyl; R²and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)_(x)—), analkyloxylalkyl linkage (—(CH₂)_(y)—O—(CH₂)_(z)—), an alkylaminoalkyllinkage (—(CH₂)_(y)NR^(a)(CH₂)_(z)) or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)—) form a ring with the carbon atom to whichthey are attached, wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a) is H,(C₁-C₆)alkyl, or phenyl; and R⁴ is unsubstituted or substituted phenylwherein the substituents are independently 1 to 5H; halo; nitro; cyano;hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁-C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino(NR^(a)CONR^(b)R^(c));mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined to form a 5- or 6-membered dioxolano (—OCH₂O—) or dioxano(—OCH₂CH₂O—) heterocyclic ring; wherein R′, Rb, and R′ are independentlyH, (C₁-C₆)alkyl, or phenyl; provided that R⁴ is not 3-nitrophenyl or4-nitrophenyl, and when R⁴ is phenyl, then R¹ is not phenyl, when R⁴ is3-chlorophenyl, then R¹ is not phenylamino, or when R⁴ is4-chlorophenyl, then R¹ is not methyl.
 13. The method of claim 12,wherein the ecdysone receptor complex is a chimeric ecdysone receptorcomplex and the DNA construct further comprises a promoter.
 14. Themethod of claim 12, wherein the subject is a plant.
 15. The method ofclaim 12, wherein the subject is a mammal.
 16. A method of modulatingthe expression of a gene in a host cell comprising the steps of: a)introducing into the host cell a gene expression modulation systemcomprising: i) a first gene expression cassette that is capable of beingexpressed in a host cell comprising a polynucleotide sequence thatencodes a first hybrid polypeptide comprising: (a) a DNA-binding domainthat recognizes a response element associated with a gene whoseexpression is to be modulated; and (b) an ecdysone receptor ligandbinding domain; ii) a second gene expression cassette that is capable ofbeing expressed in the host cell comprising a polynucleotide sequencethat encodes a second hybrid polypeptide comprising: (a) atransactivation domain; and (b) a chimeric retinoid X receptor ligandbinding domain; and iii) a third gene expression cassette that iscapable of being expressed in a host cell comprising a polynucleotidesequence comprising: (a) a response element recognized by theDNA-binding domain of the first hybrid polypeptide; (b) a promoter thatis activated by the transactivation domain of the second hybridpolypeptide; and (c) a gene whose expression is to be modulated; and b)introducing into the host cell a ligand of the formula:

wherein X and X′ are independently O or S; R¹ is a) H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, or benzyloxy; b) unsubstituted or substituted phenylwherein the substituents are independently 1 to 5 H; halo; nitro; cyano;hydroxy; amino (—NR^(a)R″); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₁-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(b)R^(c));mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacentpositions on the phenyl ring are substituted with alkoxy groups, thesegroups, together with the carbon atoms to which they are attached, maybe joined as a linkage (—OCH₂O—) or (OCH₂CH₂O—) to form a 5- or6-membered dioxolano or dioxano heterocyclic ring; c) unsubstituted orsubstituted naphthyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; d) unsubstituted orsubstituted benzothiophene-2-yl, benzothiophene-3-yl, benzofuran-2-yl,or benzofuran-3-yl wherein the substituents are independently 1 to 3halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)); e) unsubstituted or substituted 2, 3,or 4-pyridyl wherein the substituents are independently 1 to 3 halo,cyano, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or(C₁-C₆)haloalkoxy; f) unsubstituted or substituted 5-memberedheterocycle selected from furyl, thiophenyl, triazolyl, pyrrolyl,isopyrrolyl, pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl,oxazolyl, or isooxazolyl wherein the substituents are independently 1 to3 halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₁-C₆)alkoxy, (C₁-C₆)haloalkoxy,carboxy, (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)), or amino (—NR^(a)R^(b)); g)aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,phenyl(C₁-C₆)alkoxy(C₁C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein thearomatic substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or h) aromatic-substituted orunsubstituted phenylamino, phenyl(C₁-C₆)alkylamino, orphenylcarbonylamino wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; whereinR^(a), R^(b), and R^(c) are independently H, (C₁-C₆)alkyl, or phenyl; R²and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)_(x)—), analkyloxylalkyl linkage (—(CH₂),O(CH₂)_(z)), an alkylaminoalkyl linkage(—(CH₂),NR^(a)(CH₂)_(z)), or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)—) form a ring with the carbon atom towhich they are attached, wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a)is H, (C₁-C₆)alkyl, or phenyl; and R⁴ is unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5H; halo; nitro;cyano; hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a));carboxamido(CONR^(a)R^(b)); amido (—NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(b)R′);mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R³); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or when two adjacent positions onthe phenyl ring are substituted with alkoxy groups, these groups,together with the carbon atoms to which they are attached, may be joinedto form a 5- or 6-membered dioxolano (—OCH₂O—) or dioxano (—OCH₂CH₂O—)heterocyclic ring; wherein R^(a), R^(b), and R^(c) are independently H,(C₁-C₆)alkyl, or phenyl; provided that R⁴ is not 3-nitrophenyl or4-nitrophenyl, and when R⁴ is phenyl, then R¹ is not phenyl, when R⁴ is3-chlorophenyl, then R¹ is not phenylamino, or when R⁴ is4-chlorophenyl, then R¹ is not methyl.
 17. A method for producing apolypeptide comprising the steps of: a) selecting a cell which issubstantially insensitive to exposure to a ligand comprising theformula:

wherein X and X′ are independently O or S; R¹ is a) H, (C₁-C₆)alkyl,(C₁-C₆)haloalkyl, (C₁-C₆)cyanoalkyl, (C₁-C₆)alkoxycarbonyl(C₁-C₆)alkyl,(C₁-C₆)alkoxy, or benzyloxy; b) unsubstituted or substituted phenylwherein the substituents are independently 1 to 5H; halo; nitro; cyano;hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₁-C₆)haloalkylcarbonyl; benzoyl; (C₁-C₆)alkoxycarbonyl;(C₁-C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy(OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido (—NR^(a)COR^(b));alkoxycarbonylamino(NR^(a)CO₂R^(b)); alkylaminocarbonylamino(—NR^(a)CONR^(b)R^(c)); mercapto; (C₁-C₆)alkylthio; (C₁-C₆)alkylsulfonyl; (C₁-C₆)alkylsulfoxido (—S(O)R^(a)); sulfamido(—SO₂NR^(a)R^(b)); or unsubstituted or substituted phenyl wherein thesubstituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, or amino; or when two adjacent positions on the phenylring are substituted with alkoxy groups, these groups, together with thecarbon atoms to which they are attached, may be joined as a linkage(—OCH₂O—) or (OCH₂CH₂O—) to form a 5- or 6-membered dioxolano or dioxanoheterocyclic ring; c) unsubstituted or substituted naphthyl wherein thesubstituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy,(C₁-C₆)alkyl, or amino; d) unsubstituted or substitutedbenzothiophene-2-yl, benzothiophene-3-yl, benzofuran-2-yl, orbenzofuran-3-yl wherein the substituents are independently 1 to 3 halo,nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy, or(C₁-C₆)alkoxycarbonyl(—CO₂R^(a)); e) unsubstituted or substituted 2, 3,or 4-pyridyl wherein the substituents are independently 1 to 3 halo,cyano, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, or(C₁-C₆)haloalkoxy; f) unsubstituted or substituted 5-memberedheterocycle selected from furyl, thiophenyl, triazolyl, pyrrolyl,isopyrrolyl, pyrazolyl, isoimidazolyl, thiazolyl, isothiazolyl,oxazolyl, or isooxazolyl wherein the substituents are independently 1 to3 halo, nitro, hydroxy, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, carboxy,(C₁-C₆)alkoxycarbonyl (—CO₂R^(a)), or unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 3 halo, nitro,(C₁-C₆)alkyl, (C₁-C₆)haloalkyl, (C₃-C₆)alkoxy, (C₁C₆)haloalkoxy,carboxy, (C₁-C₄)alkoxycarbonyl (—CO₂R^(a)), or amino (—NR^(a)R^(b)); g)aromatic-substituted or unsubstituted phenyl(C₁-C₆)alkyl,phenyl(C₁-C₆)alkoxy(C₁-C₆)alkyl, or phenoxy(C₁-C₆)alkyl wherein thearomatic substituents are independently 1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; or h) aromatic-substituted orunsubstituted phenylamino, phenyl(C₁-C₆)alkylamino, orphenylcarbonylamino wherein the aromatic substituents are independently1 to 3 halo, nitro, (C₁-C₆)alkoxy, (C₁-C₆)alkyl, or amino; whereinR^(a), R^(b), and R^(c) are independently H, (C₃-C₆)alkyl, or phenyl; R²and R³ are independently H, (C₁-C₆)alkyl, (C₁-C₆)haloalkyl,(C₁-C₆)cyanoalkyl, (C₁-C₆)hydroxyalkyl, (C₁-C₆)alkoxy(C₁-C₆)alkyl,phenyl, or together as an alkane linkage (—(CH₂)_(x)—), analkyloxylalkyl linkage (—(CH₂),O(CH₂)z-), an alkylaminoalkyl linkage(—(CH₂)_(y)NR^(a)(CH₂)_(z)—), or an alkylbenzoalkyl linkage(—(CH₂)_(y)-1-benzo-2-(CH₂)_(z)) form a ring with the carbon atom towhich they are attached, wherein x=3 to 7, y=1 to 3, z=1 to 3, and R^(a)is H, (C₁-C₆)alkyl, or phenyl; and R⁴ is unsubstituted or substitutedphenyl wherein the substituents are independently 1 to 5H; halo; nitro;cyano; hydroxy; amino (—NR^(a)R^(b)); (C₁-C₆)alkyl; (C₁-C₆)haloalkyl;(C₁-C₆)cyanoalkyl; (C₁-C₆)hydroxyalkyl; (C₁-C₆)alkoxy; phenoxy;(C₁-C₆)haloalkoxy; (C₁-C₆)alkoxy(C₁-C₆)alkyl;(C₁-C₆)alkoxy(C₁-C₆)alkoxy; (C₁-C₆)alkanoyloxy(C₁-C₆)alkyl;(C₂-C₆)alkenyl optionally substituted with halo, cyano, (C₁-C₄)alkyl, or(C₁-C₄)alkoxy; (C₂-C₆)alkynyl optionally substituted with halo or(C₁-C₄)alkyl; formyl; carboxy; (C₁-C₆)alkylcarbonyl;(C₃-C₆)haloalkylcarbonyl; benzoyl; (C₁-C₆)alkoxycarbonyl;(C₁C₆)haloalkoxycarbonyl; (C₁-C₆)alkanoyloxy (—OCOR^(a)); carboxamido(—CONR^(a)R^(b)); amido(NR^(a)COR^(b)); alkoxycarbonylamino(—NR^(a)CO₂R^(b)); alkylaminocarbonylamino (—NR^(a)CONR^(b)R′);mercapto; (C₁-C₆)alkylthio; (C₁-C₆) alkylsulfonyl; (C₁-C₆)alkylsulfoxido(—S(O)R^(a)); sulfamido (—SO₂NR^(a)R^(b)); or unsubstituted orsubstituted phenyl wherein the substituents are independently 1 to 3halo, nitro, (C₁-C₆) alkoxy, (C₁-C₆)alkyl, or amino; or when twoadjacent positions on the phenyl ring are substituted with alkoxygroups, these groups, together with the carbon atoms to which they areattached, may be joined to form a 5- or 6-membered dioxolano (—OCH₂O—)or dioxano (—OCH₂CH₂O—) heterocyclic ring; wherein R^(a), R^(b), andR^(c) are independently H, (C₁-C₆)alkyl, or phenyl; provided that R⁴ isnot 3-nitrophenyl or 4-nitrophenyl, and when R⁴ is phenyl, then R¹ isnot phenyl, when R⁴ is 3-chlorophenyl, then R¹ is not phenylamino, orwhen R⁴ is 4-chlorophenyl, then R¹ is not methyl; b) introducing intothe cell: 1) a DNA construct comprising: i) an exogenous gene encodingthe polypeptide; and ii) a response element;  wherein the gene is underthe control of the response element; and 2) an ecdysone receptor complexcomprising: i) a DNA binding domain; ii) a binding domain for theligand; and iii) a transactivation domain; and c) exposing the cell tothe ligand.